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- Structural analysis of the interaction between Hsp90 and the tumor suppressor protein p53
- Nat Struct Mol Biol 18(10):1086-1093 (2011)
Nature Structural & Molecular Biology | Article Structural analysis of the interaction between Hsp90 and the tumor suppressor protein p53 * Franz Hagn1, 2, 4, 5 * Stephan Lagleder1, 2, 5 * Marco Retzlaff1, 4, 5 * Julia Rohrberg1 * Oliver Demmer1, 2 * Klaus Richter1 * Johannes Buchner1 * Horst Kessler1, 2, 3 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1086–1093Year published:(2011)DOI:doi:10.1038/nsmb.2114Received29 October 2010Accepted01 July 2011Published online04 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In eukaryotes, the essential dimeric molecular chaperone Hsp90 is required for the activation and maturation of specific substrates such as steroid hormone receptors, tyrosine kinases and transcription factors. Hsp90 is involved in the establishment of cancer and has become an attractive target for drug design. Here we present a structural characterization of the complex between Hsp90 and the tumor suppressor p53, a key mediator of apoptosis whose structural integrity is crucial for cell-cycle control. Using biophysical methods, we show that the human p53 DNA-binding domain interacts with multiple domains of yeast Hsp90. p53 binds to the Hsp90 C-terminal domain in its native-like state in a charge-dependent manner, but it also associates weakly with binding sites in the middle and the N-terminal domains. The fine-tuned interplay between several Hsp90 domains provides the interactions required for efficient chaperoning of p53. View full text Figures at a glance * Figure 1: Interaction between Hsp90 and p53 fragments. () Domain structure and oligomeric state of Hsp90 and p53. () Kd values for the interaction between various Hsp90 constructs and ligand-bound states and the p53-DBD obtained by fluorescence polarization (FP) experiments. () FP titration with fluorescein (FAM)-labeled p53-DBD and Hsp90 domains. () Competition between full-length (FL) Hsp90 and Hsp90-MD for FAM-p53-DBD binding shows an IC50 value of 70 μM for the MD–p53-DBD interaction. () Affinities between Hsp90 C-terminal peptides and p53-DBD. () The Hsp90–p53-DBD complex can be dissociated by p53 consensus DNA (con2x5, DNA containing two p53 consensus binding sites). (,,) Error bars, s.d. * Figure 2: NMR analysis of the binding of the p53-DBD to the individual domains of Hsp90. () 2D [15N,1H]-HSQC and TROSY spectra of the Hsp90-NTD, Hsp90-MD and Hsp90-CTD (from left to right) show high signal dispersion and well-resolved resonances. () Detailed view of some characteristic regions in the 2D [15N,1H] spectra of the isotope-labeled Hsp90 domains before (black) and after (red) addition of the p53-DBD. () CSP values mapped onto the structure of full-length Hsp90 (PDB: 2cg9). * Figure 3: NMR analysis of the binding of single Hsp90 domains to the p53-DBD. () Left, 15N HSQC sections of the p53-DBD in presence (red) and absence (black) of Hsp90-NTD in complex with AMP-PNP. CSPs occur mainly at helix 2 and loop 1 within p53-DBD. Right, CSPs mapped onto the structure of the p53-DBD. () Left, sections of p53-DBD spectra with (red contours) and without (black contours) the Hsp90-MD. Right, CSPs mapped onto the structure of p53. In contrast to the NTD, Hsp90-MD affects almost the entire p53-DBD DNA-binding interface as well as some residues within the β-barrel structure. () Left, chemical-shift perturbation of the p53-DBD by the Hsp90-CTD (black and red 15N TROSY spectra). The shift differences are much more pronounced than for the other domains and there is specific binding to the last helix (H2) and loop 1 (L1) of the p53-DBD (right). * Figure 4: Structural model of the Hsp90-MD–p53-DBD complex. () A negatively charged surface stretch in the MD mediates binding to the DNA-binding site in p53. () The interaction between the p53-DBD and its consensus DNA shows similar charge and surface properties as with Hsp90. () Paramagnetic relaxation enhancement within the p53-DBD in complex with spin-labeled Hsp90-MD variants (S282C or S456C). () Model of the Hsp90-MD–p53-DBD complex obtained with CSP and paramagnetic relaxation enhancement data. () Fluorescence polarization competition assay where the wild-type (wt) Hsp90-MD and the E412K and E415K variants dissociate the preformed Hsp90–p53-DBD complex, respectively. Bar diagrams indicate the obtained IC50 values. Error bars, s.d. () Analytical ultracentrifugation with fluorescein-labeled p53-DBD and various concentrations of Hsp90-MD wt or Hsp90-MD E415K. A shift to higher s20,w values is indicative of complex formation. * Figure 5: Structural analysis of the Hsp90-CTD–p53-DBD interaction. () Ribbon representation of Hsp90-CTD with the position of the spin labels indicated. () Paramagnetic relaxation enhancement within the p53-DBD in the presence of the spin-labeled Hsp90-CTD single-cysteine variants. () Structural model of the Hsp90-CTD peptide–p53-DBD complex obtained with NMR data–driven docking. The contacts between both proteins are mediated by a stretch of negatively charged residues within the Hsp90 C-terminal tail (side chains shown for clarity) and the positively charged DNA-binding interface of the p53-DBD consisting mainly of loop 1 (L1) and helix 2 (H2). The p53-DBD is shown as Coulomb surface, where red color represents negative and blue color positive charge, and the double arrow indicates that the C-terminal end of this Hsp90 peptide is very flexible, as proven by {1H}-15N heteronuclear NOE experiments (Supplementary Fig. 4d). * Figure 6: Chaperone activities of Hsp90 variants. () Ribbon representation of full-length Hsp90 with the location of the mutated residues indicated. () Thermal aggregation assay of the p53-DBD alone (dark gray symbols) and in presence of an equal amount of wild-type (wt) Hsp90 (blue) and the indicated variants (red and orange symbols). () In vitro translation assay with the p53-DBD and the Hsp90Δ30 and E412K variants at 10 or 70 μM concentration. The precipitate after synthesis was loaded onto a gel and the amount of p53-DBD was analyzed by densitometry. At least three experiments were done for the estimation of s.d. () Stimulation of ATPase activity of Hsp90 wt in presence of Aha1 and with or without the p53-DBD. Error bars (,), s.d. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Biological Magnetic Resonance Data Bank * 16279 Protein Data Bank * 2cg9 * 1hk7 * 2fej * 2cg9 * 1hk7 * 2fej Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Franz Hagn, * Stephan Lagleder & * Marco Retzlaff Affiliations * Center for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, Garching, Germany. * Franz Hagn, * Stephan Lagleder, * Marco Retzlaff, * Julia Rohrberg, * Oliver Demmer, * Klaus Richter, * Johannes Buchner & * Horst Kessler * Institute for Advanced Study, Technische Universität München, Garching, Germany. * Franz Hagn, * Stephan Lagleder, * Oliver Demmer & * Horst Kessler * Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. * Horst Kessler * Present addresses: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA (F.H.); Department of Biological Sciences and BioX Program, Stanford University, Stanford, California, USA (M.R.). * Franz Hagn & * Marco Retzlaff Contributions F.H., S.L. and M.R. designed research, performed experiments, analyzed data and wrote the paper. J.R., O.D. and K.R. performed research and analyzed data. J.B. and H.K. designed research and wrote the paper. F.H. and S.L. carried out protein expression, interaction studies, resonance assignment, NMR interaction and docking studies. M.R. performed protein expression, interaction studies and activity assays. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Horst Kessler or * Johannes Buchner Author Details * Franz Hagn Search for this author in: * NPG journals * PubMed * Google Scholar * Stephan Lagleder Search for this author in: * NPG journals * PubMed * Google Scholar * Marco Retzlaff Search for this author in: * NPG journals * PubMed * Google Scholar * Julia Rohrberg Search for this author in: * NPG journals * PubMed * Google Scholar * Oliver Demmer Search for this author in: * NPG journals * PubMed * Google Scholar * Klaus Richter Search for this author in: * NPG journals * PubMed * Google Scholar * Johannes Buchner Contact Johannes Buchner Search for this author in: * NPG journals * PubMed * Google Scholar * Horst Kessler Contact Horst Kessler Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–6 and Supplementary Table 1 Additional data Entities in this article * ATP-dependent molecular chaperone HSP82 HSP82 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Cellular tumor antigen p53 TP53 Homo sapiens * View in UniProt * View in Entrez Gene * Heat shock protein HSP 90-alpha HSP90AA1 Homo sapiens * View in UniProt * View in Entrez Gene * Hsp90 co-chaperone AHA1 AHA1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Bcl-2-like protein 1 BCL2L1 Homo sapiens * View in UniProt * View in Entrez Gene * Chaperone protein htpG htpG Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Hsp90 co-chaperone Cdc37 CDC37 Homo sapiens * View in UniProt * View in Entrez Gene * Cyclin-dependent kinase 4 CDK4 Homo sapiens * View in UniProt * View in Entrez Gene * Apoptosis-stimulating of p53 protein 2 TP53BP2 Homo sapiens * View in UniProt * View in Entrez Gene * Heat shock protein STI1 STI1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene - Competition between ADAR and RNAi pathways for an extensive class of RNA targets
- Nat Struct Mol Biol 18(10):1094-1101 (2011)
Nature Structural & Molecular Biology | Article Competition between ADAR and RNAi pathways for an extensive class of RNA targets * Diane Wu1 * Ayelet T Lamm2 * Andrew Z Fire1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1094–1101Year published:(2011)DOI:doi:10.1038/nsmb.2129Received12 April 2011Accepted28 July 2011Published online11 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Adenosine deaminases that act on RNAs (ADARs) interact with double-stranded RNAs, deaminating adenosines to inosines. Previous studies of Caenorhabditis elegans indicated an antagonistic interaction between ADAR and RNAi machineries, with ADAR defects suppressed upon additional knockout of RNAi. This suggests a pool of common RNA substrates capable of engaging both pathways. To define and characterize such substrates, we examined small RNA and mRNA populations of ADAR mutants and identified a distinct set of loci from which RNAi-dependent short RNAs are markedly upregulated. At these same loci, we observed populations of multiply edited transcripts, supporting a specific role for ADARs in preventing access to the RNAi pathway for an extensive population of dsRNAs. Characterization of these loci revealed a substantial overlap with noncoding and intergenic regions, suggesting that the landscape of ADAR targets may extend beyond previously annotated classes of transcripts. View full text Figures at a glance * Figure 1: Diverse consequences of dsRNA formation. Figure shows an arbitrary dsRNA (or hairpin) with a number of possible downstream processes. Left, RNA interference. (1) In the current model of RNAi, RDE-4 is involved in the recruitment of DCR-1 following recognition of a double-stranded RNAi trigger, resulting in its cleavage. (2) siRNA duplexes produced by Dicer have a characteristic structure with 5′-monophosphate, 3′-hydroxyl RNA termini for each strand and a 2-nt 3′ overhang in the duplex. (3) These cleaved duplexes subsequently program the Argonaute factor RDE-1 to recognize cognate mRNAs. (4) Following RDE-1 interaction, target mRNAs serve as templates for the transcription of a pool of 'secondary' siRNAs (short antisense transcripts templated from targeted mRNAs that carry a 5′-triphosphate (ppp 5′) terminus). Secondary siRNAs (magenta) are produced by one of two cellular RdRP enzymes: rrf-1 (somatic tissue) and ego-1 (germline). The RNAi process results in efficient and rapid loss of the pool of cognate ! mRNAs. (5) ADARs also target double-stranded RNA in vivo, converting a subset of adenines to inosine by deamination and resulting in the unwinding of the dsRNA or in other potential consequences, including alterations in mRNA stability, localization, translation and engagement in other RNA-based machineries such as RNAi42, 43. Genetic evidence in C. elegans suggests that ADAR and RNAi pathways compete for a population of substrates36. * Figure 2: Small RNA accumulation at the F07B7 histone locus in the absence of ADAR activity. An exemplary region spanning 2.6 kb (overlapping the F07B7.10 and F07B7.4 regions of C. elegans chromosome V) is shown. () Identified coding regions and conservation are diagrammed as University of California Santa Cruz (UCSC) Genome Browser tracks (C. elegans genome version WS190)56. F07B7.10 encodes an H2A histone and F07B7.4 encodes an H2B histone. Direction of transcription is depicted by arrowheads. (,) Small RNAs mapping to this region from N2 () and adr-1adr-2 animals (). Each colored rectangle represents up to ten instances of a distinct small RNA sequence per five million sequenced small RNAs. Small RNAs aligning to the (+) strand are drawn above the line, and those aligning to the (−) strand are drawn below the line. Overall numbers of aligned reads for the wild-type and adr mutant datasets in this example were 9.2 million and 10.1 million, respectively, (Supplementary Data 1) with comparable representation of miRNAs, 21U RNAs (a class of 21-nt RNAs that begin wi! th uridine) and endogenous siRNAs in the two samples. Additional examples of small RNA coverage are shown in Supplementary Figure 4. Colors indicate sizes. * Figure 3: Characteristics of ADAR-modulated RNA loci. () Histogram showing the distribution of sizes of ARLs. ARLs ranging from 100 bp to 9 kb were detected. () Venn diagram showing overlap between ARLs detected at embryo and L4 stages, based on small RNA enrichment (P < 0.05) of adr-1adr-2 animals over wild-type levels at embryo and L4 larval stages, respectively. Most ARLs are represented at both stages. (,) Overlap between ARLs and genomic repeats and features. () As shown, 82% of detected ARLs overlap annotated inverted repeats, whereas 67% overlap transposons. Fewer ARLs (18) overlap transcripts alone. A few ARLs (23) do not overlap any annotation assayed. () Partitioning of ARL annotations among annotated transcripts. Each ARL is divided into 100-bp segments, which are then indexed to the annotated genome56. Overlaps with 5′ UTR, coding, 3′ UTR, introns, miRNA and pseudogenes are then tallied, with segments overlapping two or more different annotation categories being split between the relevant classes. * Figure 4: Dependence of ARL small RNAs on the RNAi machinery. Distribution of small RNA abundances (23- to 24-nt in length only) for all 100-bp windows contained in ARLs. Small RNA values are shown as counts per million total reads aligned. Counts for each small RNA alignment were normalized to the total number of distinct genomic alignments of the associated sequence read. (–) Distribution of small RNA counts over ARLs was calculated for each of the N2 (blue) (), adr-1adr-2 (red) (), adr-1adr-2rde-4 (purple) () and adr-1adr-2rde-1 (orange) () animals. Graphs in this figure aggregate L4 and embryo data (individual distributions for L4 and embryo comparisons show a comparable difference; data not shown). Regions with high small RNA counts in wild-type animals also have comparable levels in adr-1adr-2 animals, as is evident when small RNA abundances for each 100-bp region are normalized to wild-type levels (Supplementary Fig. 2c). * Figure 5: A substantial class of additional ARL-associated siRNAs are evident in 5′ phosphate–independent capture and sequencing. () UCSC Genome Browser map (C. elegans genome version WS190) showing an ARL in the intergenic region between genes F39E9.1 (split into F39E9.1 and F39E9.22 in WS215) and Y46D2A.2 (split into Y46D2A.2 and Y46D2A.5 in WS215), overlapping the last exon of both genes. Direction of transcription is depicted by arrows. () Populations of 5′ phosphorylated small RNAs. A substantial increase in small RNA accumulation in adr-1adr-2 animals over wild-type levels can be seen at both the embryo and L4 stages. () Populations of small RNAs that have been exposed at the 5′ end by sequential treatment with alkaline phosphatase followed by polynucleotide kinase. Small RNAs accumulate (with size preference for 21- to 22-nt and a distinct strand preference) in adr-1adr-2 animals over wild-type levels at both transcribed loci overlapping the ARL. * Figure 6: Accumulation of a second population of siRNAs in cis to ARLs. () Genome-wide characterization of antisense siRNAs isolated using a 5′ phosphate–independent capture protocol. Secondary siRNAs (antisense, 20- to 23-nt) were tallied over each transcript. Transcripts overlapping ARLs are colored in brown, orange and yellow, according to the size of the total overlap (one, two, or three or more 100-bp segments, respectively). Genes that overlapped ARLs by at least three 100-bp segments and that showed a minimum six-fold increase of secondary siRNAs in adr-1adr-2 samples were deemed 'ARL-affected transcripts' and were considered as potential beneficiaries of ADAR-RNAi competition (without ADAR, they would be subject to populations of siRNAs produced by the RNAi machinery). () Effects of RNAi mutants on the secondary siRNA levels of ARL-affected transcripts. Antisense siRNA counts for ARL-affected transcripts were normalized to their respective wild-type levels. The top row (blue) denotes a separate biological preparation of wild-type ani! mals at an earlier L4 stage (2 h earlier at 20 °C). Secondary siRNA levels return to wild-type levels in adr-1adr-2rde-4 and adr-1adr-2rde-1 triple mutants. Gene-by-gene comparisons of expression-changes between different mutant backgrounds are depicted in Supplementary Figure 5. * Figure 7: A-to-G changes in mRNA show unique enrichment for ARL, inverted repeat and transposon regions. Nucleotide changes were assayed using RNA-seq data from both wild-type and adr-1adr-2 animals, using a stringent set of criteria for candidate editing sites. Sites that matched these criteria and that were absent in the adr-1adr-2 sample were reported as putative ADAR editing events. In addition to the RNA-seq analysis of embryo and L4 animals described herein, a wild-type RNA-seq dataset from L4 larvae prepared in an independent study57 was used as a replicate. (–) Distribution of genomic annotations for each class of editing event reveal enriched A-to-G edits. Edits are enriched for ADAR-modulated RNA loci as defined from small RNA sequences (), transposon regions () and regions with inverted repeat structure () but not for annotated transcribed regions (). Dashed lines indicate the fraction of editing events expected to overlap each annotation based on a random distribution across the genome (calculated using a Monte Carlo simulation as described in Online Methods). Err! or bars denote one s.d. (three samples). No other class of editing events detected show similar enrichment patterns. Within ARLs, the distribution of annotations overlapped by A-to-G editing sites is consistent: 89% of edits occur in transposon regions and 78% occur in inverted repeats (data not shown). Note that the distributions over separate annotations are not mutually exclusive (in other words, an editing site may overlap both a transposon and an inverted repeat). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Genetics, Stanford University School of Medicine, Stanford, California, USA. * Diane Wu & * Andrew Z Fire * Department of Pathology, Stanford University School of Medicine, Stanford, California, USA. * Ayelet T Lamm & * Andrew Z Fire Contributions D.W. and A.Z.F. designed experiments and wrote the paper. D.W. prepared samples, created small RNA libraries and analyzed the data. A.T.L. created mRNA libraries and participated in discussions. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Andrew Z Fire Author Details * Diane Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Ayelet T Lamm Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew Z Fire Contact Andrew Z Fire Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (7M) Supplementary Figures 1–7 and Supplementary Methods Excel files * Supplementary Data 1 (33K) Sample description * Supplementary Data 2 (123K) ADAR-affected sRNA expression over ARLs * Supplementary Data 3 (25K) Annotation enrichment of ARLs * Supplementary Data 4 (651K) Secondary siRNA expression for all genes * Supplementary Data 5 (573K) Genome-wide annotation of putative A-to-I editing * Supplementary Data 6 (78K) Statistics and raw data from Sanger sequencing Additional data Entities in this article * 5-hydroxytryptamine receptor 2C Htr2c Mus musculus * View in UniProt * View in Entrez Gene * Potassium voltage-gated channel subfamily A member 1 Kcna1 Mus musculus * View in UniProt * View in Entrez Gene * Double-stranded RNA-specific adenosine deaminase Adar Mus musculus * View in UniProt * View in Entrez Gene * Double-stranded RNA-specific editase Adar Adar Drosophila melanogaster * View in UniProt * View in Entrez Gene * Adenosine Deaminase that acts on RNA adr-1 Caenorhabditis elegans * View in Entrez Gene * Probable double-stranded RNA-specific adenosine deaminase adr-2 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * RNA interference promoting factor RDE-1 rde-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * RNA interference promoting factor rde-4 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Endoribonuclease dcr-1 dcr-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Putative uncharacterized protein Y46D2A.2 Y46D2A.2 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * RRF-1 rrf-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * RNA-directed RNA polymerase related EGO-1 ego-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Histone H2A his-51 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Histone H2B 2 his-52 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Putative uncharacterized protein F39E9.1 F39E9.1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene - Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site
- Nat Struct Mol Biol 18(10):1102-1108 (2011)
Nature Structural & Molecular Biology | Article Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site * Moran Grossman1, 2, 6 * Benjamin Born3, 6 * Matthias Heyden3, 6 * Dmitry Tworowski1 * Gregg B Fields4, 5 * Irit Sagi1, 2, 6 * Martina Havenith3, 6 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1102–1108Year published:(2011)DOI:doi:10.1038/nsmb.2120Received01 November 2010Accepted07 July 2011Published online18 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediat! ed by the catalytic metal ion and the enzyme active site. View full text Figures at a glance * Figure 1: Schematic illustration of the experimental setup to measure structural kinetics and solvation dynamics of metalloenzymes in real time. () Human MT1-MMP (gray, catalytic domain, residues 114–291) surrounded by water molecules (sticks) and bound to a peptide substrate (cyan). The catalytic zinc ion is shown as a sphere (orange). () In the enzyme active site, the zinc ion is coordinated to three histidine residues (in sticks) and to a water molecule. The active site structural kinetics are probed by transient kinetic analysis of the hydrolysis of a fluorogenic peptide using a stopped-flow device. The enzyme and the peptide substrate are rapidly mixed and the change in fluorescence intensity is detected with milliseconds time resolution. At representative times, the solution is rapidly freeze-quenched and probed by XAS at the zinc K-edge energy, providing the metal ion oxidation state, coordination number, bond distances and disorder. () Changes in the collective water network dynamics of the enzyme-substrate mixture during enzymatic turnover are probed by THz pulses in the KITA experiment. At selected times ! after mixing, the net THz absorption is recorded. * Figure 2: Real-time spectroscopic analysis of metalloenzyme (MT1-MMP) catalysis of the peptide substrate Mca-PLGL(Dnp)AR. () Pre-steady state analysis showing the fluorescence emission (○) after mixing MT1-MMP and the fluorogenic substrate Mca-PLGL(Dnp)AR in a stopped-flow apparatus (λexcitation (λex) = 340 nm, λemission (λem) > 380 nm). Fluorescence intensity is displayed in arbitrary units (AU). A 60 ms kinetic lag phase precedes a kinetic burst (dashed line and arrow). Inset, intact substrate (left) and products of substrate hydrolysis (right). () KITA (○) showing the time-resolved transmitted electric field amplitude of the THz pulse for the enzyme-substrate mixture (Emix) as well as for the buffer (Ebuffer) under experimental conditions identical to those for . Inset, KITA of buffer-buffer mixing. () Increased formation of the active intermediate, correlated with changes in solvent dynamics. The relative fractions of the pentacovalent active intermediate (in columns) were determined by principal component analysis on XAS analysis of the MT1-MMP catalytic site during substrate turno! ver. Error bars are from the residuals of the principal component analysis. Inset, structure of the pentacoordinated Michaelis complex. () Active site charge fluctuations during substrate binding and turnover (in columns) derived from the shifts in zinc K-edge absorption energies ± s.e.m. Inset, representative charge fluctuations at the catalytic zinc ion of MT1-MMP during turnover. * Figure 3: Analysis of time-dependent X-ray absorption spectra. The data are presented in the form of Fourier transform spectra to provide the radial distribution of the atoms within the first and second coordination shell of the catalytic zinc ion in MT1-MMP, where R is the zinc-ligand bond distance in angstroms (○). The shape and amplitude of the Fourier transform peaks are directly related to the type and number of amino acid residues that are bound to the zinc ion. The increase in the first shell amplitude is correlated with an increase in coordination number (curved black arrow). * Figure 4: Changes in hydration dynamics are mostly associated with Michaelis complex formation. () Comparison between the transient kinetic traces of hydrolysis of 6-mer peptide substrate (○) versus a 'poor' substrate (blue triangles) of the sequence Mca-Arg-Pro-Lys-Pro-Ala-Nva-Trp-Met-Lys(Dnp)-NH2 (Nva = norvaline) by MT1-MMP. The kinetic analysis (shown in log scale for clarity) features an extended lag phase of 100 ms. () An extended exponential decay in net THz absorption of 140 ms is observed during hydrolysis of the 'poor' peptide substrate by MT1-MMP, synchronized with the kinetic lag phase (blue triangles). () The interaction between MT1-MMP and its endogenous inhibitor TIMP-2 was monitored by the increase in the MT1-MMP intrinsic fluorescence (⋄). Complex formation is achieved within 400 ms. () The change in solvent dynamics during MT1-MMP–TIMP-2 interaction probed by KITA is not found to be synchronized with complex formation. The time constant, when assuming an exponential decrease, is on the order of 7 s. The net change of THz absorption is on the ord! er of 0.8%. AU, arbitrary units. * Figure 5: Gradient of coupled protein-water motions. () Changes in hydration dynamics upon Michaelis complex formation. Shown are the averaged hydrogen bond lifetimes τHB of bulk water (red line; distance to closest protein atom >12 Å), water molecules solvating MT1-MMP (magenta line; distance to closest enzyme atom <6 Å), water molecules solvating the bound substrate (blue line; distance to closest substrate atom <6 Å), and water molecules solvating the zinc ion of the free catalytic site before substrate binding (cyan line; distance to zinc ion <6 Å). () Water dynamics gradient at catalytic site and response upon complex formation. Selected water molecules from each hydration water layer are presented as spheres for clarity and are color coded as in . The enzyme surface is shown in gray and the catalytic zinc ion as a yellow sphere. Left: a steep gradient of water motions (from strongly retarded water molecules (blue) to bulk (red)) is detected in the free enzyme before substrate binding to the catalytic zinc ion. Middl! e: initially, substrate molecules (white) nonspecifically bind to the enzyme surface. Hydration water molecules of the substrate show dynamics similar to enzyme hydration water. The overall gradient in the hydrogen bond dynamics at the active site is proposed to assist peptide binding through a remote water-retardation mechanism mediated by the charged metalloenzyme active site. Right: after substrate binding to the zinc ion, a smooth gradient of water hydrogen bond dynamics motions is generated while the water molecules solvating the substrate are slowed down (blue). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 1BUV * 1BUV Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Moran Grossman, * Benjamin Born, * Matthias Heyden, * Irit Sagi & * Martina Havenith Affiliations * Department of Structural Biology, The Weizmann Institute of Science, Rehovot, Israel. * Moran Grossman, * Dmitry Tworowski & * Irit Sagi * Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel. * Moran Grossman & * Irit Sagi * Lehrstuhl für Physikalische Chemie II, Ruhr-Universität Bochum, Bochum, Germany. * Benjamin Born, * Matthias Heyden & * Martina Havenith * Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas, USA. * Gregg B Fields * The Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida, USA. * Gregg B Fields Contributions I.S. and M.Ha. are equal contributors and designed the experiments, analyzed the data and wrote the manuscript. M.G., B.B. and M.He. are equal contributors and conducted the research, analyzed the data and wrote the manuscript. D.T. constructed the enzyme-substrate docking model. G.B.F. provided the substrates. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Irit Sagi or * Martina Havenith Author Details * Moran Grossman Search for this author in: * NPG journals * PubMed * Google Scholar * Benjamin Born Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Heyden Search for this author in: * NPG journals * PubMed * Google Scholar * Dmitry Tworowski Search for this author in: * NPG journals * PubMed * Google Scholar * Gregg B Fields Search for this author in: * NPG journals * PubMed * Google Scholar * Irit Sagi Contact Irit Sagi Search for this author in: * NPG journals * PubMed * Google Scholar * Martina Havenith Contact Martina Havenith Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Methods Additional data Entities in this article * Matrix metalloproteinase-14 MMP14 Homo sapiens * View in UniProt * View in Entrez Gene * Metalloproteinase inhibitor 2 TIMP2 Homo sapiens * View in UniProt * View in Entrez Gene - Structure of collagenase G reveals a chew-and-digest mechanism of bacterial collagenolysis
- Nat Struct Mol Biol 18(10):1109-1114 (2011)
Nature Structural & Molecular Biology | Article Structure of collagenase G reveals a chew-and-digest mechanism of bacterial collagenolysis * Ulrich Eckhard1 * Esther Schönauer1 * Dorota Nüss1 * Hans Brandstetter1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1109–1114Year published:(2011)DOI:doi:10.1038/nsmb.2127Received01 March 2011Accepted21 July 2011Published online25 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Collagen constitutes one-third of body protein in humans, reflecting its extensive role in health and disease. Of similar importance, therefore, are the idiosyncratic proteases that have evolved for collagen remodeling. The most efficient collagenases are those that enable clostridial bacteria to colonize their host tissues; but despite intense study, the structural and mechanistic basis of these enzymes has remained elusive. Here we present the crystal structure of collagenase G from Clostridium histolyticum at 2.55-Å resolution. By combining the structural data with enzymatic and mutagenesis studies, we derive a conformational two-state model of bacterial collagenolysis, in which recognition and unraveling of collagen microfibrils into triple helices, as well as unwinding of the triple helices, are driven by collagenase opening and closing. View full text Figures at a glance * Figure 1: Domain organization and architecture of ColG. () Schematic of the domain organization of ColG, with functional annotation. The catalytic Zn2+ ion (yellow dot) and the catalytic residues (red stars) within the peptidase domain are indicated. The hatched domains (prepro-peptide and collagen-binding domains CBD-a and CBD-b) are not present in the crystal. () Ribbon representation of the collagenase module, colored as in . The position of the PKD-like domain (yellow ribbon) at the rear of the peptidase domain is indicated in surface representation, reflecting a positional variance of up to 10 Å. The saddle-shaped collagenase is composed of an activator and a peptidase domain. The catalytic Zn2+ and the catalytic residues are shown in ball-and-stick representation. The seat of the saddle is formed by the distorted four-helix bundle, represented by four cylinders, and completed by the glycine-rich hinge, shown in light green. () Full-length model of ColG in complex with a collagen microfibril. The collagenase module (ribbon ! representation, colored as in ) is shown bound to a modeled collagen microfibril. Accessory domains are shown as surface representation. The two CBDs (orange) were not present in the crystal but were oriented and positioned to satisfy the biochemically derived binding data, including the inferred binding epitopes. Arrow indicates direction of collagenase processivity. * Figure 2: The activator domain is necessary for the degradation of collagen. () Relative peptidolytic activities of ColG variants against FALGPA, normalized to the wild-type (WT) protein. Tyr119–Gly790, Tyr119–Asn880, Tyr119–Gly1001, Lys396–Lys1118 are segments comprising the indicated residues; Lys396–Lys1118 lacks the N-terminal activator domain. Δ(Gly389–Val397) is a full-length protein with the indicated deletion, which cuts out the glycine-rich hinge. Data are shown as mean ± s.d. (n = 6). () Relative collagenolytic activities of indicated variants against fluorescein-labeled collagen type I, given as relative initial velocities, normalized to WT. Chymotrypsin serves as a negative control, indicating that the collagen substrate was not denatured. Data are shown as mean ± s.d. (n = 36). * Figure 3: Mapping the peptidase active site. () Ribbon representation of the active site. Three protein residues (His523, His527 and Glu555, shown in red) and a water molecule (blue sphere) tetrahedrally coordinate the catalytic Zn2+ (yellow sphere). Glu524 hydrogen-bonds to the water molecule. The nonprimed substrate-recognition sites are formed by the antiparallel edge strand extending from Gly494 to Glu498 (orange). The S1′ oxyanion site is formed by the amides of Gly493–Gly494 (bright orange). () Accessible surface representation of the ColG active site containing the inhibitor isoamyl-phosphonyl-Gly-Pro-Ala in stick representation, oriented and colored as in . The inhibitor is overlaid with the experimental 2Fo – Fc omit electron density, contoured at 1 σ over the mean (light gray). Substrate recognition is governed at the nonprimed sites by the antiparallel edge strand, by the catalytic Zn2+ (dark red with yellow sphere) and by the secondary S1′ oxyanion pocket. A prominent wall delimits the S3′ site a! nd explains the tripeptidyl-carboxypeptidase activity of ColG. * Figure 4: Unified processing model of triple-helical and microfibrillar collagen. () A collagen triple helix (green) initially docks to the peptidase domain of collagenase (colored as in Fig. 1). In the open state, the activator (dark blue) cannot interact with the substrate, and no hydrolysis occurs. () Step 2, closed conformation, showing the activator HEAT repeats interacting with the triple helix, which is a prerequisite for collagen hydrolysis. () Step 3, semi-open conformation, allowing for exchange and processive degradation of all three α-chains31, 32. Once the triple helix is completely cleaved, the collagenase can relax back to the open ground-state conformation, as found in our crystals, and thus complete the catalytic cycle. () Collagenase with a docked collagen microfibril (gray; based on PDB 4CLG). The microfibril typically consists of five triple-helical molecules19; the triple helix analogous to that in is indicated in green. () Step 2, closed conformation with all triple helices but one (green) being expelled from the collagenase. The mi! crofibril 'lesion' caused by the stripping of the triple-helix is indicated in red. () Step 3, semi-open conformation allowing for complete processing of the triple helix, indicated in green. Next, the collagenase will relax back to the open state and only then allow the remaining part of the microfibril to enter the collagenase. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2Y3U * 2Y50 * 2Y6I * 2Y72 * 2Y3U * 2Y50 * 2Y6I * 2Y72 Referenced accessions Protein Data Bank * 4CLG * 4CLG Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Division of Structural Biology, Department of Molecular Biology, University of Salzburg, Salzburg, Austria. * Ulrich Eckhard, * Esther Schönauer, * Dorota Nüss & * Hans Brandstetter Contributions U.E. performed experiments (protein production, enzymological measurements, crystallization, X-ray data collection and structure determination), analyzed data and prepared the manuscript; E.S. performed experiments (enzymological measurements), analyzed data and prepared the manuscript; D.N. performed experiments (crystal harvesting and X-ray data collection); H.B. devised the project, helped with structure solution, analyzed data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hans Brandstetter Author Details * Ulrich Eckhard Search for this author in: * NPG journals * PubMed * Google Scholar * Esther Schönauer Search for this author in: * NPG journals * PubMed * Google Scholar * Dorota Nüss Search for this author in: * NPG journals * PubMed * Google Scholar * Hans Brandstetter Contact Hans Brandstetter Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (737K) Supplementary Figures 1 and 2 Additional data Entities in this article * Collagenase Clostridium histolyticum * View in UniProt * Leukotriene A-4 hydrolase LTA4H Homo sapiens * View in UniProt * View in Entrez Gene * Tricorn protease-interacting factor F3 Ta0815 Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165) * View in UniProt * View in Entrez Gene * Thermolysin Bacillus thermoproteolyticus * View in UniProt * ColH protein Clostridium histolyticum * View in UniProt - Spliceosome assembly is coupled to RNA polymerase II dynamics at the 3′ end of human genes
- Nat Struct Mol Biol 18(10):1115-1123 (2011)
Nature Structural & Molecular Biology | Article Spliceosome assembly is coupled to RNA polymerase II dynamics at the 3′ end of human genes * Sandra Bento Martins1, 3 * José Rino1, 3 * Teresa Carvalho1 * Célia Carvalho1 * Minoru Yoshida2 * Jasmim Mona Klose1 * Sérgio Fernandes de Almeida1 * Maria Carmo-Fonseca1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1115–1123Year published:(2011)DOI:doi:10.1038/nsmb.2124Received24 January 2011Accepted19 July 2011Published online04 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In the nucleus of higher eukaryotes, maturation of mRNA precursors involves an orderly sequence of transcription-coupled interdependent steps. Transcription is well known to influence splicing, but how splicing may affect transcription remains unclear. Here we show that a splicing mutation that prevents recruitment of spliceosomal snRNPs to nascent transcripts causes co-transcriptional retention of unprocessed RNAs that remain associated with polymerases stalled predominantly at the 3′ end of the gene. In contrast, treatment with spliceostatin A, which allows early spliceosome formation but destabilizes subsequent assembly of the catalytic complex, abolishes 3′ end pausing of polymerases and induces leakage of unspliced transcripts to the nucleoplasm. Taken together, the data suggest that recruitment of splicing factors and correct assembly of the spliceosome are coupled to transcription termination, and this might ensure a proofreading mechanism that slows down release ! of unprocessed transcripts from the transcription site. View full text Figures at a glance * Figure 1: Imaging β-globin transcription sites in vivo. () Schematic representation of the β-globin expression plasmid (pTRE-βWT-MS2exon) used for stable integration into the genome of human U2OS cells. Expression is controlled by seven copies of the tetracycline response element (TRE mod (×7)). In the presence of tetracycline derivative doxycycline (Dox), the tetracycline-controlled transactivator (rtTA) activates transcription from the minimal cytomegalovirus promoter (Pmin CMV). The human β-globin gene sequence encompasses 1,827 nucleotides past the poly(A) site. Dimers of MS2 protein fused to mCherry recognize MS2-binding stem-loops introduced in exon 3, allowing the transcribed RNA to be visualized. The numbers indicate exon and intron size in nucleotides (nts). ATG: start codon. () Clone U2OS-βWT#9 was transiently transfected with a plasmid expressing mCherry-MS2 and imaged alive before (−Dox) and after (+Dox) incubation with doxycycline for 24 h. (–) Detection of β-globin transcripts by FISH. Nontransfected U2OS ! Tet-On cells () and clone U2OS-βWT#9 (,) were hybridized with a probe complementary to β-globin RNA (red staining). Where indicated, cells were incubated with doxycycline for 24 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue staining). () Identification of β-globin transcription sites by FISH. Simultaneous detection of DNA and RNA was carried out in U2OS-βWT#9 cells incubated with doxycycline for 24 h. The RNA probe (green staining) hybridizes to β-globin transcripts, and the DNA probe (complementary to the pTRE vector, red staining) reveals the site of integration of transgenes in the genome. A representative double-hybridization merged image is shown. The enlarged inset shows a transcription site with the corresponding intensity line scans. Scale bars indicate 5 μm. * Figure 2: Characterization of U2OS-βWT cells. () Relative copy number of transgene integrations. Genomic DNA was isolated and analyzed by semiquantitative PCR using specific pairs of primers (represented by arrows). In U2OS-βWT#9 cells, four additional amplification cycles of the endogenous HBB gene are required to obtain a level of PCR product similar to that of the transgene. Thus, the transgene is 24 (16) times more abundant than the endogenous β-globin gene. Because U2OS cells contain two copies of the endogenous gene, we conclude that clone U2OS-βWT#9 contains ~32 copies of integrated transgenes. () Analysis of β-globin transcripts. Transcription of β-globin was induced by doxycycline for 24 h. Total RNA was isolated from induced (+Dox) and non-induced (−Dox) cells. Semiquantitative RT-PCR analysis was done using primers specific for the spliced (S) RNA. Molecular size markers (bp) are indicated on the left. (,) Analysis of β-globin protein. Total lysates were collected from cells before (−Dox) and after ! (+Dox) induction with doxycycline for 24 h and analyzed by immunoblotting using antibodies directed against β-globin (HBB), β-actin and GAPDH. To estimate the half-life of transgenic β-globin protein, lysates were collected 0, 1, 2 and 4 h after treatment with cycloheximide (CHX). Molecular weight markers (kDa) are indicated on the left. * Figure 3: Imaging β-globin transcription dynamics. (,) Representative images of live U2OS-βWT cells that express RNAPII-GFP and MS2 protein-mCherry, before () and after () incubation with doxycycline. () FRAP experiments. Unprocessed images are shown. Insets show corresponding post-processed fluorescence-normalized images of the region of interest in pseudocolor. Scale bars indicate 5 μm. () Fluorescence recovery of mCherry-MS2 was measured at the transcription site or in a nucleoplasmic region distant from the transgene locus. The total number of cells analyzed was 25 and 15, respectively. () FRAP curves for mCherry-MS2 (red) and RNAPII-GFP (green). The total number of cells analyzed was 25. () FRAP experiments were conducted using the same bleaching settings and number of time points as in panel but with a longer time interval between each image. A total of five cells were analyzed. In all panels, the curves correspond to a pool of at least three independent experiments and the error bars represent s.d. The t50% and tmax! values represent the mean time required to reach half-maximal and maximal recovery, respectively. () Chromatin immunoprecipitation analysis was done in doxycycline-induced cells with antibodies to total RNAPII. The schematic diagram on the top indicates the gene regions amplified by primer sets (exons and past poly(A) region (pp(A))). Data are expressed as fold enrichment relative to the control intergenic region and normalized to RNAPII loading on the first exon. Histograms depict mean and s.d. from at least three independent experiments. * Figure 4: Effect of intron 2 deletion on β-globin RNA splicing and polymerase dynamics. () Schematic representation of the mutant variant of β-globin that completely lacks the second intron (βIVS1) and contains six MS2-binding stem-loops introduced in exon 3. RNA was amplified with the indicated primers (represented by arrows). The spliced (S) and unspliced (U) products are indicated on the right. Molecular size markers (bp) are indicated on the left. (,) FRAP curves for mCherry-MS2 () and RNAPII-GFP () measured in cells stably transfected with either the wild-type or mutant variant of the β-globin gene. A total of ten mutant cells were analyzed. In all panels, the curves correspond to a pool of at least three independent experiments and the error bars represent s.d. The t50% and tmax values represent the mean time required to reach half-maximal and maximal recovery, respectively. () Chromatin immunoprecipitation with antibodies to total RNAPII. The schematic diagram on the top indicates the gene regions amplified by primer sets (exons and past poly(A) regio! n (pp(A))). Data are expressed as fold enrichment relative to the control intergenic region and normalized to RNAPII loading on the first exon. () Total RNA was reverse transcribed and qrtPCR amplified using primer pairs across exon 1 and past the poly(A) site. The ratios of abundance of the two qrt-PCR products was calculated and plotted in the histogram. All histograms depict mean and s.d. for three independent experiments. The asterisk denotes statistically significant differences (Student's t-test, P < 0.05). * Figure 5: Effect of SSA on β-globin RNA splicing and polymerase dynamics. U2OS-βWT cells were induced with doxycycline and either untreated (−SSA) or treated (+SSA) with 100 ng ml−1 SSA for 24 h. () Total RNA was isolated, reversed transcribed with random primers and PCR amplified with the indicated primers (represented by arrows). The spliced (S) and unspliced (U) products are illustrated on the right. Molecular size markers (bp) are indicated on the left. () FRAP curves for mCherry-MS2 in treated (black) and untreated (red) cells. The total number of cells analyzed was 28 and 25, respectively. () FRAP curves for RNAPII-GFP in treated (black) and untreated (green) cells. The total number of cells analyzed was 28 and 25, respectively. (–) FRAP curves from cells that were either untreated or transfected with GL2 (RNAi GL2; n = 16) (), or transfected with control (RNAi GL2; n = 16) or SAP130 (RNAi SAP130; n = 10) siRNAs (). () FRAP curves for RNAPII-GFP in a pool of U2OS cells stably transfected with β-globin cDNA (βcDNA) that were either u! ntreated (−SSA; n = 12) or treated (+SSA; n = 13). Note that fluorescence recovery is faster in the β-globin cDNA compared to the wild-type gene (shown in ), consistent with the reduced size of the cDNA (403 fewer nucleotides than the wild-type gene). All curves correspond to a pool of at least three independent experiments. Error bars represent s.d. * Figure 6: SSA reduces RNAPII density downstream of the poly(A) site in both β-globin and endogenous genes. (–) U2OS-βWT cells were induced with doxycycline and either not treated (−SSA) or treated (+SSA) with 100 ng ml−1 SSA for 24 h. Chromatin immunoprecipitation analysis was done with antibodies to total RNAPII (), Ser2P RNAPII () and CDK9 (). The schematic diagram at the top indicates the gene regions amplified by primer sets (exons and past poly(A) region, pp(A)). Data are expressed as fold enrichment relative to the control intergenic region. () Total RNA was isolated, reverse transcribed with random primers and PCR amplified using the indicated primer pairs. The amount of amplified product obtained with each primer set was estimated by qrt-PCR. Ratios of abundance relative to exon1 values were calculated and plotted. () U2OS cells were either not treated (−SSA) or treated (+SSA) with 100 ng ml−1 SSA for 24 h. Chromatin immunoprecipitation was done with antibodies to total RNAPII on MYC, GAPDH and p21 genes. Transcription of the p21 gene was induced by Nutlin-3a, ! as described36. The schematic diagrams on the top indicate the gene regions amplified by primer sets. Data are expressed as fold enrichment relative to the control intergenic region. All histograms depict mean and s.d. from at least three independent experiments. The asterisk denotes statistically significant differences (Student's t-test, P < 0.05). * Figure 7: Coupling spliceosome assembly to leakage of unspliced transcripts to the nucleoplasm. (–) RNA transcribed from βWT and βIVS1 transgenes was visualized by FISH. Cells were double-labeled for β-globin RNA with the indicated probes (red staining) and total DNA (blue staining). (,) Cells were not treated (−DRB) or treated (+DRB) with the transcriptional inhibitor DRB for 5 min. () Cells were not treated (−SSA) or treated (+SSA) with SSA for 24 h. Scale bars indicate 10 μm. (,) Cells were double-labeled for the transgene site of integration in the genome by DNA FISH with the indicated probe (red staining) and for protein by indirect immunofluorescence (green staining), using antibodies specific for the indicated spliceosomal proteins. () Cells expressing βIVS1 transcripts. () Cells expressing βWT transcripts, before (−SSA) and after (+SSA) treatment with SSA for 24 h. Representative double-hybridization merged images are shown. The enlarged insets show a transcription site with the corresponding intensity line scans depicted on top. The color of the ! lines in the graphics corresponds to the color of the detection of DNA and protein in the cell. Scale bar indicates 5 μm. * Figure 8: Schematic model illustrating the main conclusions from this study. During transcription of the wild-type β-globin gene, dissociation of polymerases from the DNA downstream of the poly(A) site is a slow process; this is reflected by transient immobilization of RNAPII-GFP molecules detected by FRAP. In the case of the βIVS1 mutant, the unspliced RNA does not recruit the U1 and U2 snRNPs and remains tethered to the DNA in association with the polymerase downstream of the poly(A) site. This is reflected by reduced recovery kinetics of both RNA and RNAPII molecules. Treatment with SSA, which allows recruitment of U1 and U2 snRNPs but blocks subsequent formation of a catalytic spliceosome, leads to faster dissociation of polymerases from wild-type β-globin DNA after the poly(A) site. In FRAP experiments, RNAPII immobilization is no longer detected and unspliced RNA is released to the nucleoplasm. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Sandra Bento Martins & * José Rino Affiliations * Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. * Sandra Bento Martins, * José Rino, * Teresa Carvalho, * Célia Carvalho, * Jasmim Mona Klose, * Sérgio Fernandes de Almeida & * Maria Carmo-Fonseca * Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Saitama, Japan. * Minoru Yoshida Contributions S.B.M. and J.R. designed and conducted the experiments and analyzed the data. J.R. was responsible for the microscopy. T.C. and C.C. generated and characterized the cell lines. J.M.K. carried out ChIP experiments and S.F.d.A. designed, conducted and analyzed biochemical experiments. M.Y. synthesized SSA. M.C.-F. conceived and supervised the project. S.B.M., J.R. and M.C.-F. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Maria Carmo-Fonseca Author Details * Sandra Bento Martins Search for this author in: * NPG journals * PubMed * Google Scholar * José Rino Search for this author in: * NPG journals * PubMed * Google Scholar * Teresa Carvalho Search for this author in: * NPG journals * PubMed * Google Scholar * Célia Carvalho Search for this author in: * NPG journals * PubMed * Google Scholar * Minoru Yoshida Search for this author in: * NPG journals * PubMed * Google Scholar * Jasmim Mona Klose Search for this author in: * NPG journals * PubMed * Google Scholar * Sérgio Fernandes de Almeida Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Carmo-Fonseca Contact Maria Carmo-Fonseca Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–6 and Supplementary Tables 1 and 2 Additional data Entities in this article * Hemoglobin subunit beta HBB Homo sapiens * View in UniProt * View in Entrez Gene * Coat protein cp Enterobacteria phage MS2 * View in UniProt * View in Entrez Gene * G1/S-specific cyclin-D1 CCND1 Homo sapiens * View in UniProt * View in Entrez Gene * Splicing factor 3B subunit 3 SF3B3 Homo sapiens * View in UniProt * View in Entrez Gene * Splicing factor 3B subunit 2 SF3B2 Homo sapiens * View in UniProt * View in Entrez Gene * Splicing factor 3B subunit 1 SF3B1 Homo sapiens * View in UniProt * View in Entrez Gene * Cyclin-dependent kinase 9 CDK9 Homo sapiens * View in UniProt * View in Entrez Gene * Cyclin-dependent kinase inhibitor 1 CDKN1A Homo sapiens * View in UniProt * View in Entrez Gene * Glyceraldehyde-3-phosphate dehydrogenase GAPDH Homo sapiens * View in UniProt * View in Entrez Gene * Myc proto-oncogene protein MYC Homo sapiens * View in UniProt * View in Entrez Gene * Splicing factor 1 SF1 Homo sapiens * View in UniProt * View in Entrez Gene * U1 small nuclear ribonucleoprotein A SNRPA Homo sapiens * View in UniProt * View in Entrez Gene * Splicing factor U2AF 65 kDa subunit U2AF2 Homo sapiens * View in UniProt * View in Entrez Gene * U2 small nuclear ribonucleoprotein B'' SNRPB2 Homo sapiens * View in UniProt * View in Entrez Gene * Beta-actin ACTB Homo sapiens * View in UniProt * View in Entrez Gene * Serine/arginine repetitive matrix protein 1 SRRM1 Homo sapiens * View in UniProt * View in Entrez Gene - The Rad50 coiled-coil domain is indispensable for Mre11 complex functions
- Nat Struct Mol Biol 18(10):1124-1131 (2011)
Nature Structural & Molecular Biology | Article The Rad50 coiled-coil domain is indispensable for Mre11 complex functions * Marcel Hohl1 * Youngho Kwon2 * Sandra Muñoz Galván3 * Xiaoyu Xue2 * Cristina Tous3 * Andrés Aguilera3 * Patrick Sung2 * John H J Petrini1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1124–1131Year published:(2011)DOI:doi:10.1038/nsmb.2116Received04 March 2011Accepted27 June 2011Published online04 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The Mre11 complex (Mre11, Rad50 and Xrs2 in Saccharomyces cerevisiae) influences diverse functions in the DNA damage response. The complex comprises the globular DNA-binding domain and the Rad50 hook domain, which are linked by a long and extended Rad50 coiled-coil domain. In this study, we constructed rad50 alleles encoding truncations of the coiled-coil domain to determine which Mre11 complex functions required the full length of the coils. These mutations abolished telomere maintenance and meiotic double-strand break (DSB) formation, and severely impaired homologous recombination, indicating a requirement for long-range action. Nonhomologous end joining, which is probably mediated by the globular domain of the Mre11 complex, was also severely impaired by alteration of the coiled-coil and hook domains, providing the first evidence of their influence on this process. These data show that functions of Mre11 complex are integrated by the coiled coils of Rad50. View full text Figures at a glance * Figure 1: Rad50 coiled-coil mutants. () Mre11 complexes in the process of bridging sisters. () Rad50 coiled-coil mutants created in this study. TEV protease recognition sites, orange and green; hook, purple. Dashed line indicates that TEV protease cleavage of Rad502TEV, Rad50sc and Rad50sc+h leads to almost identical Rad50 cleavage products lacking the hook domain. Mre11-interacting interface of Rad50 near the Rad50 ATPase core25, 26, 27 is indicated. aa, amino acids; pI, isoelectric point. () Rad502TEV primary protein structure with 2TEV recognition sequences at residues 573 and 829 on either side of the hook. Expected molecular masses of protease cleavage fragments (f1–f5) are indicated. () Cleavage of the Rad50 hook domain by TEV protease in rad502TEV cells. Wild-type (WT) Rad50 (153 kDa), full-length Rad502TEV (157 kDa; fl) and Rad502TEV cleavage products were visualized by Rad50 immunoprecipitation and Rad50 western blot. Expression of the Myc epitope–tagged TEV protease was monitored in extracts by we! stern blotting with Myc antisera (bottom). () In vitro TEV cleavage of WT Rad50 and Rad502TEV proteins. Full-length Rad50 was immunoprecipitated with Rad50 antisera, cleaved with recombinant TEV protease and analyzed by western blotting. () Rad50 pull-down assay of 3× HA-tagged Rad50 proteins after TEV protease induction in vivo. Rad502TEV C-terminal cleavage fragments were detected by western blot with antibody to HA. The HA3 tag adds ~5 kDa to the molecular mass. Rad50 cleavage fragments in are labeled as in . * Figure 2: Mre11 complex integrity and DNA association are intact without the hook and most of the coiled coil. () Coomassie blue stain of reconstituted Rad502TEV-containing Mre11 complexes with and without TEV cleavage. () TEV-cleaved Rad502TEV interacts with Mre11. Indicated complexes containing His6-Mre11 were captured with Ni-NTA agarose beads with and without TEV cleavage. Supernatant (s), wash (w) and SDS eluate (e) were analyzed by SDS-PAGE. The f2 hook fragment remained noncovalently associated after TEV cleavage under conditions used. () Integrity of the Mre11 complex in wild-type (WT) Rad50 and rad50coils. Rad50-HA-tagged (lanes 2–11), Flag-Mre11 (lanes 1–6) or untagged Mre11 (lanes 7–11) were immunoprecipitated (IP) and analyzed by western blotting with the indicated antisera. Arrow, expected migration level of the Rad50nc+h protein. () DNA binding of Rad502TEV alone and in complex. Top, EMSA in presence of ATP with 0 (lanes 1, 8 and 15), 50 (lanes 2, 9 and 16), 100 (lanes 3, 10 and 17), 150 (lanes 4, 11 and 18) and 200 nM Rad50 (lanes 5, 12 and 19); untreated WT (lef! t), untreated Rad502TEV (middle) and TEV treated Rad502TEV (right). Rad50 (200 nM) was incubated in binding buffer without ATP (lanes 6, 13 and 20) or treated with SDS and proteinase K (lanes 7, 14 and 21). Bound (b) and unbound (u) DNA was separated under native conditions. Rad50 and Rad502TEV alone (bottom left) or in complex with Mre11 and Xrs2 (MRX, bottom right). Representative gel pictures are in Supplementary Figure 2b. * Figure 3: rad50 hook and coiled-coil mutants are defective in meiotic DSB formation and telomere maintenance. () Diploid wild-type (WT) SK1 and rad50sc+h cells were cultivated in sporulation medium for 2 d and stained with DAPI. () Meiotic DSB formation at the HIS4-LEU2 meiotic hotspot. Cells were cultivated for 0, 4, 8 and 12 h in sporulation medium (SPM) and meiotic DSB formation was detected by Southern blot with a hotspot-specific probe. Arrows, migration levels of the two major Spo11-cleavage fragments (3.7 and 6.0 kilobases, kb) and a minor one (~10 kb). () Telomere length after 0, 30 and 60 generations of growth at 30 °C or 23 °C. Genomic DNA was digested with PstI and telomeres were visualized by Southern blot using a telomere-specific probe. () Plating efficiency after 10 and 50 generations of growth at 30 °C. Error bars correspond to s.d. of three experiments. * Figure 4: The Rad50 hook and coiled-coil domains are required for cell survival in the presence of MMS. Cell survival on plates containing indicated concentrations of MMS without (−TEV; glucose) or with TEV protease expression (+TEV; galactose). All strains contain the TEV expression cassette, except one labeled –GAL-TEV. Plates were incubated for 4 d at 30 °C. MMS sensitivity at different incubation temperatures (23 °C and 37 °C) and a quantitative assessment of the MMS sensitivity are in Supplementary Figure 1a–c. * Figure 5: SCR is promoted by the Rad50 hook and coiled-coil domain. () pRS316-TINV SCR plasmid and products, unequal sister chromatid exchange (SCE) and intrachromatid recombination (ICR). The 4.7-kb band specific for SCE and the 2.9-kb band for SCE and ICR can be visualized after XhoI/SpeI digestion in Southern blots using a LEU2 probe7, 31. () Kinetics of DSB formation and repair. Top, HO induction for 0, 2, 4 and 6 h without (−TEV; no GAL-TEV) or with TEV protease expression (+TEV). Ratio of 4.7-kb band and total plasmid was used to calculate SCE. Bottom, HO induction over 24 h (0, 3, 6, 9, 12 and 24 h) without TEV expression. WT, wild type. () Assessment of spontaneous ade2 heteroallelic mitotic recombination. Diploid ade2-n/ade2-l-Scel, WT RAD50 or rad50 mutant colonies were replated on plates with limiting concentration of adenine. White sectors (ADE2) within red colonies (ade2) were scored (see photographs here and in Supplementary Fig. 4b). Percentage of colonies containing 0, 1–2, 3–4, >4 and >10 white sector(s) and average nu! mbers of sectors per colony. Error bars correspond to s.d. of 5–7 diploids analyzed. * Figure 6: NHEJ in rad50coils. Mean values are indicated.() Plasmid-based NHEJ assays (Online Methods). () Survival frequency of strains with chronic HO induction. Repair junctions were analyzed; numbers of small insertions or deletions are indicated. Sequences are in Supplementary Figure 5a. () Top, cell survival upon chronic HO induction without or with concomitant TEV expression. Bottom, repair junctions of rad502TEV survivors (without or with TEV) were analyzed qPCR, quantitative PCR. Sequences are listed in Supplementary Figure 5c. () NHEJ repair kinetics after acute (2 h) HO induction were determined by qPCR. Cells were cultivated for another 1.5, 3 or 5 h in glucose medium after HO induction and repair was monitored with primers flanking the HO site. () Cell survival and NHEJ repair events after acute HO DSB induction. Top, cell survival after 2 h HO induction. Bottom, numbers of imprecise NHEJ repair events versus total numbers of HO junctions sequenced. rad50nc+h was assessed only in one experime! nt. Error bars refer to s.d. of at least three determinations. * Figure 7: Distal domain alterations and potential effects on the globular domain. Two lobes of the heterotetrameric Mre11 complex globular DNA-binding domain, from which two extended coiled coils protrude. Globular domain orientation, arrows; zigzag lines, flexible regions. To account for the effects of rad50coils and rad502TEV on NHEJ, we propose that coiled-coil shortening and TEV-mediated hook cleavage perturb the coils in two nonexclusive manners. Axial rotation of the coils, causing misalignment of the globular domain, is envisioned in both rad50coils and rad502TEV, and altered flexibility is envisioned in rad50coils mutants. In these scenarios, axial rotations may cause distortion of the globular domain that alters the disposition of DNA ends held within it. Alterations in coiled-coil flexibility may disrupt the juxtaposition of two ends. Both outcomes could alter the efficiency and junctional sequences of NHEJ. Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Chromosome Biology, Memorial Sloan‐Kettering Cancer Center, New York, New York, USA. * Marcel Hohl & * John H J Petrini * Yale University School of Medicine, New Haven, Connecticut, USA. * Youngho Kwon, * Xiaoyu Xue & * Patrick Sung * Centro Andaluz de Biología Molecular y Medicina Regenerativa, Universidad de Sevilla, Sevilla, Spain. * Sandra Muñoz Galván, * Cristina Tous & * Andrés Aguilera Contributions M.H., Y.K., X.X., S.M.G. and C.T. carried out experiments. J.H.J.P. designed research in consultation with P.S. and A.A. J.H.J.P. and M.H. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * John H J Petrini Author Details * Marcel Hohl Search for this author in: * NPG journals * PubMed * Google Scholar * Youngho Kwon Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra Muñoz Galván Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaoyu Xue Search for this author in: * NPG journals * PubMed * Google Scholar * Cristina Tous Search for this author in: * NPG journals * PubMed * Google Scholar * Andrés Aguilera Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick Sung Search for this author in: * NPG journals * PubMed * Google Scholar * John H J Petrini Contact John H J Petrini Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods Additional data Entities in this article * DNA repair protein RAD50 RAD50 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Double-strand break repair protein MRE11 MRE11 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA repair protein XRS2 XRS2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Nibrin NBN Homo sapiens * View in UniProt * View in Entrez Gene * DNA repair protein RAD50 RAD50 Homo sapiens * View in UniProt * View in Entrez Gene * Meiosis-specific protein SPO11 SPO11 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Histidine biosynthesis trifunctional protein HIS4 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * 3-isopropylmalate dehydrogenase LEU2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA endonuclease SAE2 SAE2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Phosphoribosylaminoimidazole carboxylase ADE2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA polymerase IV POL4 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * ATP-dependent DNA helicase II subunit 2 YKU80 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Ligase-interacting factor 1 LIF1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DNA repair protein RAD5 RAD5 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * N-(5'-phosphoribosyl)anthranilate isomerase TRP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Repressible acid phosphatase PHO5 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Tubulin beta chain TUB2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Actin ACT1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene - Defects in RNA quality control factors reveal RNAi-independent nucleation of heterochromatin
- Nat Struct Mol Biol 18(10):1132-1138 (2011)
Nature Structural & Molecular Biology | Article Defects in RNA quality control factors reveal RNAi-independent nucleation of heterochromatin * Francisca E Reyes-Turcu1, 3 * Ke Zhang1, 3 * Martin Zofall1 * Eesin Chen1, 2 * Shiv I S Grewal1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1132–1138Year published:(2011)DOI:doi:10.1038/nsmb.2122Received04 January 2011Accepted08 July 2011Published online04 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Heterochromatin assembly at Schizosaccharomyces pombe centromeres involves a self-reinforcing loop mechanism wherein chromatin-bound RNAi factors facilitate targeting of Clr4–Rik1 methyltransferase. However, the initial nucleation of heterochromatin has remained elusive. We show that cells lacking Mlo3, a protein involved in mRNP biogenesis and RNA quality control, assemble functional heterochromatin in RNAi-deficient cells. Heterochromatin restoration is linked to RNA surveillance because loss of Mlo3-associated TRAMP also rescues heterochromatin defects of RNAi mutants. mlo3Δ, which causes accumulation of bidirectional repeat-transcripts, restores Rik1 enrichment at repeats and triggers de novo heterochromatin formation in the absence of RNAi. RNAi-independent heterochromatin nucleation occurs at selected euchromatic loci that show upregulation of antisense RNAs in mlo3Δ cells. We find that the exosome RNA degradation machinery acts parallel to RNAi to promote heteroch! romatin formation at centromeres. These results suggest that RNAi-independent mechanisms exploit transcription and non-coding RNAs to nucleate heterochromatin. View full text Figures at a glance * Figure 1: mlo3Δ restores functional heterochromatin at centromeres in the ago1Δ mutant. () mlo3Δ suppresses the silencing defect at otr1::ura4+ in ago1Δ cells. Location of ura4+ reporter inserted within the pericentromeric region is shown. Shown are the results of serial dilutions of the indicated strains spotted onto non-selective 5-fluoroacetic acid (−FOA) or counter-selective FOA-containing (+FOA) media to assay ura4+ expression. () mlo3Δ decreases RNAPII occupancy at centromeric repeats in ago1Δ cells. RNAPII levels were determined by ChIP combined with microarray (ChIp-chip) using Ser2phospho RNAPII antibody and plotted in alignment with the right pericentromeric region of cen2. () mlo3Δ restores centrometric heterochromatin in ago1Δ cells. ChIP analysis of H3K9me and Swi6 at otr1::ura4+. DNA isolated from immunoprecipitated chromatin or whole-cell crude extracts (WCE) was analyzed by PCR. Relative fold enrichments depicting the ratios of signals at otr1::ura4+ locus relative to the euchromatic ura4DS/E (DS/E) locus, between ChIP and WCE, are shown! underneath each lane. () mlo3Δ restores heterochromatin at dg repeats in ago1Δ cells. H3K9me and Swi6 enrichments at dg repeats relative to leu1 were determined by ChIP. () mlo3Δ suppresses TBZ sensitivity and restores cohesin localization at centromeres in the ago1Δ mutant. Localization of cohesin subunit Rad21 (Rad21-HA) at otr1::ura4+ was assessed by ChIP (bottom). () Mlo3 interacts with dh transcripts. Interaction of Mlo3 with the fbp1 transcripts, used as a control, and dh transcripts was determined by RNA immunoprecipitation. RNA isolated from immunoprecipitated Mlo3-Flag (Flag RIP) or whole cell extract (input) was analyzed by RT-PCR. –RT, no reverse transcription. * Figure 2: tfs1Δ restores centromeric heterochromatin in ago1Δ cells. () tfs1Δ renders cells sensitive to 6-AU. () tfs1Δ suppresses the silencing defect at otr1::ura4+ in ago1Δ cells. Shown are the results of serial dilutions of WT and mutant strains carrying mat1-Msmt0 mating-type allele that were spotted onto the indicated media to assay otr1::ura4+ expression. () tfs1Δ decreases RNAPII occupancy at centromeric repeats in ago1Δ cells. RNAPII occupancy at dh/dg repeats in centromere 2 was determined by ChIP-chip using Ser2 phospho RNAPII antibody. () tfs1Δ restores heterochromatin at otr1::ura4+ in ago1Δ cells. Relative fold enrichments of H3K9me and Swi6 at otr1::ura4+ were determined by ChIP. () tfs1Δ restores heterochromatin at dg repeats in ago1Δ cells. H3K9me and Swi6 enrichments at dg repeats were determined by ChIP. () tfs1Δ suppresses TBZ sensitivity of ago1Δ cells. () tfs1Δ restores cohesin localization to centromeres. Localization of Rad21-HA at otr1::ura4+ was assessed by ChIP. * Figure 3: tfs1Δ and mlo3Δ differentially suppress heterochromatin defects in clr3Δ ago1Δ double mutant cells. mlo3Δ, but not tfs1Δ, restores H3K9me at centromeres in clr3Δ ago1Δ cells. (,) Relative fold enrichments of H3K9me at otr1::ura4+ were determined by ChIP. (,) H3K9me levels across pericentromeric domains of cen2 as determined by ChIP-chip. * Figure 4: Loss of RNA surveillance factor TRAMP restores centromeric heterochromatin in ago1Δ cells. (,) cid14Δ restores heterochromatin at otr1::ura4+ and dg repeats in ago1Δ cells. H3K9me levels at otr1::ura4+ and dg were determined by ChIP. () cid14Δ suppresses centromeric silencing defects in ago1Δ cells. RT-PCR analysis of dg and dh transcripts in the indicated strains is shown. fbp1 transcripts were assayed as an amplification control. * Figure 5: Rrp6 acts parallel to RNAi to mediate heterochromatin formation and silencing at centromeres. () Cid14 and Rrp6 differentially affect dh expression in ago1Δ cells. Northern blot analysis of dh transcripts in WT and mutant cells. rRNA was used as a loading control. () rrp6Δ and ago1Δ cause cumulative increase in dg and dh transcript levels. RT-PCR analysis of dg and dh transcript levels in the indicated strains is shown. fbp1 transcripts were assayed as an amplification control. () Loss of Rrp6 in ago1Δ cells severely affects H3K9me at centromeres. H3K9me enrichment at otr1::ura4+ and dg repeats were determined by ChIP. () Deletions of rrp6 and ago1 cause cumulative loss in H3K9me across pericentromeric domains, as determined by ChIP-chip. * Figure 6: mlo3Δ restores Rik1 enrichment at centromeric repeats and triggers de novo heterochromatin formation in the absence of RNAi. () mlo3Δ induces establishment of heterochromatin at centromeres in the absence of Ago1. Indicated mutant strains were transformed with a plasmid containing clr4+ gene. Levels of H3K9me were assayed by ChIP at otr1::ura4+ and dg repeats in the indicated strains. () mlo3Δ causes increased accumulation of bidirectional transcripts and restores Rik1 ChIP enrichment at cenH in ago1Δ mutant. Schematic representation indicating the location of cenH and primers used (black bar) is shown (upper). IR-R and IR-L boundary elements surrounding the silent mat locus are shown. ChIP analysis of Rik1-myc at cenH (left). RT-PCR analysis of cenH transcripts (right). fbp1 transcripts were assayed as an amplification control. 'Rev' and 'For' indicate reverse and forward transcripts, respectively. * Figure 7: RNAi-independent heterochromatin formation occurs at euchromatic loci in mlo3Δ cells. () Appearance of H3K9me in mlo3Δ cells correlates with the accumulation of antisense transcripts. Relative fold enrichment of H3K9me was determined by ChIP-chip in WT and mutant strains. Red bars indicate locations of primers used for RT-PCR analysis shown in . () RT-PCR analysis of sense and antisense at indicated loci in WT and mlo3Δ cells. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Francisca E Reyes-Turcu & * Ke Zhang Affiliations * Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA. * Francisca E Reyes-Turcu, * Ke Zhang, * Martin Zofall, * Eesin Chen & * Shiv I S Grewal * Present address: Department of Biochemistry, National University of Singapore, Singapore. * Eesin Chen Contributions F.E.R.-T., K.Z. and S.I.S.G. designed the research. K.Z., F.E.R.-T. and M.Z. conducted the experiments. E.C. contributed the reagents. F.E.R.-T., K.Z. and S.I.S.G. analyzed the data. F.E.R.-T. and S.I.S.G. wrote the paper. F.E.R.-T., K.Z. and S.I.S.G. edited the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Shiv I S Grewal Author Details * Francisca E Reyes-Turcu Search for this author in: * NPG journals * PubMed * Google Scholar * Ke Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Zofall Search for this author in: * NPG journals * PubMed * Google Scholar * Eesin Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Shiv I S Grewal Contact Shiv I S Grewal Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (10M) Supplementary Figures 1–8 and Supplementary Methods Additional data Entities in this article * Histone-lysine N-methyltransferase, H3 lysine-9 specific clr4 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Chromatin modification-related protein rik1 rik1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * mRNA export protein mlo3 mlo3 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Protein argonaute ago1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Protein Dicer dcr1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * RNA-dependent RNA polymerase 1 rdp1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Chromo domain-containing protein 1 chp1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * RNA-induced transcriptional silencing complex protein tas3 tas3 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Histone-lysine N-methyltransferase SUV39H1 SUV39H1 Homo sapiens * View in UniProt * View in Entrez Gene * Chromo domain-containing protein 2 chp2 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Chromatin-associated protein swi6 swi6 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Histone chaperone cia1 cia1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Histone deacetylase clr6 clr6 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Poly(A) RNA polymerase cid14 cid14 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * RNA annealing protein YRA1 YRA1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * THO complex subunit 4 THOC4 Homo sapiens * View in UniProt * View in Entrez Gene * Orotidine 5'-phosphate decarboxylase ura4 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Fructose-1,6-bisphosphatase fbp1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Transcription elongation factor A protein 1 TCEA1 Homo sapiens * View in UniProt * View in Entrez Gene * Histone deacetylase clr3 clr3 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Transcription elongation factor S-II tfs1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Probable ubiquitin carboxyl-terminal hydrolase 3 ubp3 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Exosome complex exonuclease rrp6 rrp6 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * 3-isopropylmalate dehydrogenase leu1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Cohesin subunit rad21 rad21 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene - Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other microRNAs
- Nat Struct Mol Biol 18(10):1139-1146 (2011)
Nature Structural & Molecular Biology | Article Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other microRNAs * David M Garcia1, 2, 3, 8 * Daehyun Baek1, 2, 3, 4, 5, 8 * Chanseok Shin1, 2, 3, 6 * George W Bell1 * Andrew Grimson1, 2, 3, 7 * David P Bartel1, 2, 3 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1139–1146Year published:(2011)DOI:doi:10.1038/nsmb.2115Received12 December 2010Accepted01 July 2011Published online11 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Most metazoan microRNAs (miRNAs) target many genes for repression, but the nematode lsy-6 miRNA is much less proficient. Here we show that the low proficiency of lsy-6 can be recapitulated in HeLa cells and that miR-23, a mammalian miRNA, also has low proficiency in these cells. Reporter results and array data indicate two properties of these miRNAs that impart low proficiency: their weak predicted seed-pairing stability (SPS) and their high target-site abundance (TA). These two properties also explain differential propensities of small interfering RNAs (siRNAs) to repress unintended targets. Using these insights, we expand the TargetScan tool for quantitatively predicting miRNA regulation (and siRNA off-targeting) to model differential miRNA (and siRNA) proficiencies, thereby improving prediction performance. We propose that siRNAs designed to have both weaker SPS and higher TA will have fewer off-targets without compromised on-target activity. View full text Figures at a glance * Figure 1: Strengthening SPS while decreasing TA imparted typical targeting proficiency to lsy-6 and miR-23 miRNAs. () Sequences of miRNAs and target sites tested in reporter assays. Each miRNA was co-transfected with reporter plasmids as a duplex designed to represent the miRNA paired with its miRNA* strand (Supplementary Fig. 1a). () Response of reporters with 3′ UTRs of predicted lsy-6 targets after co-transfection with lsy-6. As a specificity control, the experiment was also done using a noncognate miRNA, miR-1 (gray bars). Geometric means are plotted relative to those of reporters in which the predicted target sites were mutated after also normalizing for the repression observed for miR-1 (gray bars). Mutant sites of this experiment were the cognate sites of Figure 2d. Error bars, third largest and third smallest values among 12 replicates from 4 independent experiments. Significant differences in repression by cognate miRNA compared to that by noncognate miRNA are indicated. () Distribution of predicted SPSs for 7-mer-m8 sites of 60 conserved nematode miRNA families36 (Supplementa! ry Data 2). Values were rounded down to the next half-integer unit. () SPS distribution for 7-mer-m8 sites of 87 conserved vertebrate miRNA families8 (Supplementary Data 2). () Distributions of predicted genome TA for 7-mer-m8 3′ UTR sites of 60 conserved nematode miRNA families (Supplementary Data 2). Values were rounded up to the next tenth of a unit. () Distributions of predicted genome TA for 7-mer-m8 3′ UTR sites of 87 conserved vertebrate miRNA families (Supplementary Data 2). () Response of reporters mutated such that their sites matched the miR-142 seed. The cognate miRNA was the miR-142lsy-6 chimera; noncognate sites were lsy-6 sites. Otherwise, as in . () As in , except showing the response to miR-142 transfection. () Response of reporters with 3′ UTRs of predicted miR-23 targets after co-transfection with miR-23a. Noncognate sites were for miR-CGCG. Otherwise, as in . () Response of reporters mutated such that their sites matched the seed of miR-CGCG, which! was co-transfected as the cognate miRNA. Noncognate sites wer! e for miR-23. Otherwise, as in . *P < 0.01, **P < 0.001, Wilcoxon rank-sum test. * Figure 2: Separating the effects of SPS and TA on miRNA targeting proficiency. () Relationship between predicted SPS and genomic TA for lsy-6 and the 59 other conserved nematode miRNAs (red squares), and all other heptamers (light blue, blue, dark blue or purple squares indicating 0, 1, 2 or 3 CpG dinucleotides within the heptamer, respectively). TA was defined as total number of canonical 7- to 8-nt sites (8-mer, 7-mer-m8 and 7-mer-A1) in annotated 3′ UTRs. SPS values were predicted using the respective 7-mer-m8 sites. () Relationship between predicted SPS and TA in human 3′ UTRs for miR-23 and the 86 other broadly conserved vertebrate miRNA families (red squares). Otherwise, as in . () Sequences of miRNAs and target sites tested in reporter assays of this figure. () Response of reporters with 3′ UTRs of predicted lsy-6 targets mutated such that their sites matched the seed of LTA-lsy-6, which was co-transfected as the cognate miRNA. Noncognate sites were for lsy-6. Otherwise, as in Figure 1b. () 2,6-di-aminopurine (DAP or D)-uracil base pair. (! ) Response of reporters used in after co-transfecting D-LTA-lsy-6 as the cognate miRNA. Otherwise, as in . () Response of reporters used in Figure 1i after co-transfecting D-miR-23a as the cognate miRNA, alongside results for miR-23a that was repeated in parallel. Otherwise, as in Figure 1i. *P < 0.01, **P < 0.001, Wilcoxon rank-sum test. * Figure 3: Impact of TA and SPS on sRNA targeting proficiency, as determined using array data. () Distribution of TAHeLa and predicted SPS for the sRNAs from the 102 array data sets analyzed in this study (orange squares) and sRNAs from data sets that passed the motif-enrichment analysis (red squares). Otherwise, plotted as in Figure 2b. () Response of expressed mRNAs with a single 3′ UTR site to the cognate sRNA, with respect to TAHeLa and predicted SPS. Fold-change values are plotted according the key to the right of each plot, comparing mRNAs with a single site of the type indicated (and no additional sites to the cognate sRNA elsewhere in the mRNA) to those with no site to the cognate sRNA; note different scales for different plots. In areas of overlap, mean values are plotted. Correlation coefficients and P values are in Table 1. () Response of expressed mRNAs with a single ORF site to the cognate sRNA, with respect to TAHeLa and predicted SPS. Otherwise, as in . () Response of mRNAs with indicated single sites when binning cognate sRNA by TAHeLa (top) or predi! cted SPS (bottom). The key indicates the data considered, with the first quartiles at top comprising data for sRNAs with the lowest TAHeLa and those at bottom comprising data for sRNAs with the strongest predicted SPS. Error bars, 95% confidence intervals. * Figure 4: Predictive performance of the context+ model, which considers miRNA or siRNA proficiency in addition to site context. () Improved predictions for mRNAs with canonical 7- to 8-nt 3′ UTR sites. Predicted interactions between mRNAs and cognate sRNA were distributed into ten equally populated bins based on total context scores generated using the model indicated (key), with the first bin comprising interactions with most favorable scores. Plotted for each bin is the mean mRNA change on the arrays (error bars, 95% confidence intervals). () Prediction of responsive interactions involving mRNAs with only 3′ UTR 6-mer sites. Otherwise, as in . () Prediction of responsive interactions involving mRNAs with at least one 8-mer ORF site but no 3′ UTR sites. Otherwise, as in . () Impact of TA and SPS on siRNA-directed knockdown of the desired target. Efficacy in luciferase activity knockdown for 2,431 siRNAs transfected into H1299 cells38. Efficacy is linearly scaled (key), with positive and negative controls having values of 0.900 and 0.354, respectively38. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * David M Garcia & * Daehyun Baek Affiliations * Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. * David M Garcia, * Daehyun Baek, * Chanseok Shin, * George W Bell, * Andrew Grimson & * David P Bartel * Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * David M Garcia, * Daehyun Baek, * Chanseok Shin, * Andrew Grimson & * David P Bartel * Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * David M Garcia, * Daehyun Baek, * Chanseok Shin, * Andrew Grimson & * David P Bartel * School of Biological Sciences, Seoul National University, Seoul, Republic of Korea. * Daehyun Baek * Bioinformatics Institute, Seoul National University, Seoul, Republic of Korea. * Daehyun Baek * Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea. * Chanseok Shin * Present address: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. * Andrew Grimson Contributions D.M.G. carried out most reporter assays and associated experiments and analyses. D.B. carried out all the computational analyses except for reporter analyses. G.W.B. implemented revisions to the TargetScan site. C.S. and A.G. carried out assays and analyses involving miR-23. D.M.G., D.B. and D.P.B. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * David P Bartel or * Daehyun Baek Author Details * David M Garcia Search for this author in: * NPG journals * PubMed * Google Scholar * Daehyun Baek Contact Daehyun Baek Search for this author in: * NPG journals * PubMed * Google Scholar * Chanseok Shin Search for this author in: * NPG journals * PubMed * Google Scholar * George W Bell Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew Grimson Search for this author in: * NPG journals * PubMed * Google Scholar * David P Bartel Contact David P Bartel Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2.2 MB) Supplementary Figures 1–5 and Supplementary Tables 1–5. Excel files * Supplementary Data 1 (25 KB) 175 microarrays analyzed in this study. * Supplementary Data 2 (23 KB) Human and C. elegans miRNA families, conserved in vertebrates and nematodes, respectively. * Supplementary Data 4 (17.5 MB) mRNA fold-change values. * Supplementary Data 5 (4 MB) Predicted SPS and TA values for all heptamers in C. elegans, human and HeLa, mouse, and D. melanogaster. Zip files * Supplementary Data 3 (18 MB) Reference mRNAs. Additional data Entities in this article * microRNA lsy-6 C32C4.6 Caenorhabditis elegans * View in Entrez Gene * Protein C27H6.9 C27H6.9 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * microRNA 142 MIR142 Homo sapiens * View in Entrez Gene * microRNA 23a MIR23A Homo sapiens * View in Entrez Gene * Protein F55G1.12 F55G1.12 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Protein T20G5.9 T20G5.9 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Protein argonaute-2 EIF2C2 Homo sapiens * View in UniProt * View in Entrez Gene * Induced by phosphate starvation1 IPS1 Arabidopsis thaliana * View in UniProt * View in Entrez Gene - Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum
- Nat Struct Mol Biol 18(10):1147-1152 (2011)
Nature Structural & Molecular Biology | Article Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum * Ethan J Greenblatt1, 2 * James A Olzmann2 * Ron R Kopito1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1147–1152Year published:(2011)DOI:doi:10.1038/nsmb.2111Received08 February 2011Accepted05 July 2011Published online11 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The degradation of misfolded secretory proteins is ultimately mediated by the ubiquitin-proteasome system in the cytoplasm, therefore endoplasmic reticulum–associated degradation (ERAD) substrates must be dislocated across the ER membrane through a process driven by the AAA ATPase p97/VCP. Derlins recruit p97/VCP and have been proposed to be part of the dislocation machinery. Here we report that Derlins are inactive members of the rhomboid family of intramembrane proteases and bind p97/VCP through C-terminal SHP boxes. Human Derlin-1 harboring mutations within the rhomboid domain stabilized mutant α-1 antitrypsin (NHK) at the cytosolic face of the ER membrane without disrupting the p97/VCP interaction. We propose that substrate interaction and p97/VCP recruitment are separate functions that are essential for dislocation and can be assigned respectively to the rhomboid domain and the C terminus of Derlin-1. These data suggest that intramembrane proteolysis and protein disl! ocation share unexpected mechanistic features. View full text Figures at a glance * Figure 1: Derlins belong to the rhomboid family. () ClustalW alignment (UniProtKB accession number indicated) of the transmembrane regions of Derlin-1 from Homo sapiens (hs, Q9BUN8), Danio rerio (dr, Q7ZVT9), Caenorhabditis elegans (ce, Q93561), Derlin-2 (hs, Q9GZP9) and Derlin-3 (hs, Q96Q80) from H. sapiens and GlpG from Shewanella trabarsenatis (st, Q0HDA1), GlpG, Yersinia frederiksenii (yf, C4SQG5), and Escherichia coli (ec, P09391). Pink, the active site dyad in GlpG, absent in Derlins. WR and GxxxG motifs are indicated by . (–) Homology model of the Derlin-1 rhomboid domain. The positions of the WR motif (), GxxxG motif () and positively charged residues on the cytoplasmic side of each transmembrane span in the homology model () are indicated. N- and C termini of Derlin-1 are located in the cytoplasm. () Representative immunofluorescence images of cells expressing N- and C-terminally tagged HA-Derlin-1-S in which the plasma membrane was permeabilized without (digitonin) or with (digitonin + Triton X-100) ER permeabi! lization. Scale bars are 10 μm. () Comparison of the four-pass topology model of Derlin-1 as inferred from the reported topology of the Saccharomyces cerevisiae ortholog Der1 (ref. 8) with the six-pass topology model of Derlin-1 as predicted by homology with GlpG. () Derlin-1 spans the ER membrane six times. Immunoblot of lysates treated with or without Endo H from cells expressing Derlin-1-S containing a glycosylation acceptor sequence inserted after the indicated residue. * Figure 2: p97/VCP binding to Derlin-1 through an SHP box is required for efficient dislocation of NHK. () Domain organization of Derlin-1-S and Derlin-2-S constructs used in this study. () Alignment of SHP boxes from the p97/VCP/Cdc48 binding proteins H. sapiensDerlin-1 and Derlin-2, S. cerevisiaeDfm1, H. SapiensUfd1 and p47 and S. cerevisiaeShp1. () The SHP box is required for Derlin-1 and Derlin-2 interactions with p97/VCP. Immunoblot of digitonin lysates and S-protein agarose–precipitated material from untransfected (UT) cells or cells expressing wild-type (WT) or mutant Derlin-1-S or Derlin-2-S lacking the SHP box (1–240 and 1–231, respectively). (–) Overexpression of Derlin-1 lacking the SHP box results in stabilization of deglycosylated forms of NHK associated with Derlin-1. () Immunoblots of digitonin lysates or S-protein agarose–precipitated material from cells coexpressing NHK-HA with the indicated Derlin-1-S variants or a GFP-S control. Deglycosylated forms of NHK-HA are indicated by arrowheads. () Immunoblot of four biological replicates similar to those ! in , except cells were lysed in SDS lysis buffer. () Quantification of the levels of deglycosylated NHK-HA in as a percentage of total NHK-HA levels. Error bars represent s.e.m. () Immunoblot of material from treated with or without Endo H. () Left panel, immunoblots of Triton X-100 lysate from cells coexpressing NHK-HA, wild-type or mutant Derlin-1 or a control protein (ΔCD4) and an shRNA targeting GFP or PNG1. Quantification of data was carried out as in . n.d., not detectable. To enable detection, Derlin-1-S and mPNG1-S were enriched by S-protein agarose precipitation. Right panel, efficacy of shRNA-mediated PNG1 knockdown. CHO, carbohydrate; AP, affinity precipitation. * Figure 3: The GxxxG motif in the Derlin-1 rhomboid domain is required for the dislocation of NHK. () Derlin-1G176V acts a dominant negative mutant by inhibiting the dislocation of NHK. Immunoblots of digitonin lysates and S-protein agarose–precipitated material from cells coexpressing NHK-HA and the indicated Derlin-1-S variant or a GFP control. Deglycosylated forms of NHK are indicated by arrowheads. () Quantification of deglycosylated NHK-HA levels in as a percentage of total NHK-HA levels. () Deglycosylated forms of NHK are exposed to the cytoplasm. Immunoblots of membranes obtained from homogenized cells coexpressing NHK-HA and Derlin-1-SG176V treated with the indicated concentrations of proteinase K and Triton X-100. The cytoplasmic C-terminal S-tag of Derlin-1-S, or luminal (SEL1L, GRP94 and BiP) markers were used as controls. () Quantification of the data from . () Disruption of NHK dislocation is specific to mutation of the Derlin-1 GxxxG motif. Immunoblots of SDS lysates from cells coexpressing NHK-HA with wild-type Derlin-1-S or the indicated GxxxG mutants (G! 176V or G180V) or mutants outside of the GxxxG motif (G29V or G147V), or a GFP control, treated with or without Endo H. () Mutation of the GxxxG motif does not disrupt homo-oligomerization of Derlin-1 or interaction with p97/VCP. Immunoblots of digitonin lysates and S-protein agarose–precipitated material from cells expressing wild-type or mutant Derlin-1-S or a GFP control. * Figure 4: The Derlin-1 WR motif is essential for the dislocation of NHK. () Expression of Derlin-1 WR motif mutants results in a reduction in NHK levels in Triton X-100 lysates. Immunoblots of Triton X-100 lysates from cells coexpressing NHK-HA and Derlin-1-SWT, the indicated Derlin-1-S WR motif mutants (Q51A, W53A, R54A or T57A), Derlin-1-S mutated at a nonconserved tryptophan (W106A), or a GFP control. () Overexpression of Derlin-1R54A results in aggregation and accumulation of deglycosylated NHK. Immunoblots of Triton X-100 soluble and insoluble material. () Mutation of the Derlin-1 WR motif does not disrupt interaction with p97/VCP. Arrowheads indicate deglycosylated NHK. (–) Expression of Derlin-1 containing mutations in the WR or GxxxG rhomboid motifs stabilizes NHK. () Immunoblot of SDS lysates from cells coexpressing NHK-HA and wild-type or mutant Derlin-1-S or a GFP control that were treated with emetine for the indicated durations. () Quantification of four independent experiments is shown. Asterisks denotes P ≤ 0.05. Error bars ind! icate s.e.m. () Derlin-1 GxxxG and WR motif mutants are stable and expressed at similar levels. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 2IC8 * 2IC8 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Biophysics Program, Stanford University, Stanford, California, USA. * Ethan J Greenblatt & * Ron R Kopito * Department of Biology, Stanford University, Stanford, California, USA. * Ethan J Greenblatt, * James A Olzmann & * Ron R Kopito Contributions E.J.G. and R.R.K. contributed to the design of all of the experiments and wrote the manuscript. E.J.G. did the experiments and analyses in Figure 1a–d,f,g; Figure 2–4; and Supplementary Figures 1, 2, 4 and 5. The microscopy experiments in Figure 1e and Supplementary Figures 3 and 6 were conducted by J.A.O. All authors contributed to the interpretation and conclusions of the experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ron R Kopito Author Details * Ethan J Greenblatt Search for this author in: * NPG journals * PubMed * Google Scholar * James A Olzmann Search for this author in: * NPG journals * PubMed * Google Scholar * Ron R Kopito Contact Ron R Kopito Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–6 Additional data Entities in this article * Derlin-1 DERL1 Homo sapiens * View in UniProt * View in Entrez Gene * Alpha-1-antitrypsin SERPINA1 Homo sapiens * View in UniProt * View in Entrez Gene * Transitional endoplasmic reticulum ATPase VCP Homo sapiens * View in UniProt * View in Entrez Gene * Unique short US11 glycoprotein Human cytomegalovirus (strain AD169) * View in UniProt * Cell division control protein 48 CDC48 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Derlin-2 DERL2 Homo sapiens * View in UniProt * View in Entrez Gene * Derlin-3 DERL3 Homo sapiens * View in UniProt * View in Entrez Gene * Degradation in the endoplasmic reticulum protein 1 DER1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * DER1-like family member protein 1 DFM1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase synoviolin SYVN1 Homo sapiens * View in UniProt * View in Entrez Gene * Protein sel-1 homolog 1 SEL1L Homo sapiens * View in UniProt * View in Entrez Gene * Rhomboid protease glpG glpG Escherichia coli (strain K12) * View in UniProt * View in Entrez Gene * Epidermal growth factor receptor EGFR Homo sapiens * View in UniProt * View in Entrez Gene * Presenilins-associated rhomboid-like protein, mitochondrial PARL Homo sapiens * View in UniProt * View in Entrez Gene * FAS-associated factor 2 FAF2 Homo sapiens * View in UniProt * View in Entrez Gene * Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase NGLY1 Homo sapiens * View in UniProt * View in Entrez Gene * Ubiquitin fusion degradation protein 1 UFD1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * 78 kDa glucose-regulated protein HSPA5 Homo sapiens * View in UniProt * View in Entrez Gene * Endoplasmin HSP90B1 Homo sapiens * View in UniProt * View in Entrez Gene * Rhomboid protein 1, mitochondrial PCP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Cytochrome c peroxidase, mitochondrial CCP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Inactive rhomboid protein 1 rho-5 Drosophila melanogaster * View in UniProt * View in Entrez Gene * Epidermal growth factor receptor Egfr Drosophila melanogaster * View in UniProt * View in Entrez Gene * Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase Ngly1 Mus musculus * View in UniProt * View in Entrez Gene * T-cell surface glycoprotein CD4 CD4 Homo sapiens * View in UniProt * View in Entrez Gene * Ubiquitin fusion degradation protein 1 homolog UFD1L Homo sapiens * View in UniProt * View in Entrez Gene * NSFL1 cofactor p47 NSFL1C Homo sapiens * View in UniProt * View in Entrez Gene * UBX domain-containing protein 1 SHP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene - Recognition of the pre-miRNA structure by Drosophila Dicer-1
- Nat Struct Mol Biol 18(10):1153-1158 (2011)
Nature Structural & Molecular Biology | Article Recognition of the pre-miRNA structure by Drosophila Dicer-1 * Akihisa Tsutsumi1, 2 * Tomoko Kawamata1 * Natsuko Izumi1 * Hervé Seitz3, 4 * Yukihide Tomari1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1153–1158Year published:(2011)DOI:doi:10.1038/nsmb.2125Received19 January 2011Accepted13 July 2011Published online18 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Drosophila melanogaster has two Dicer proteins with specialized functions. Dicer-1 liberates miRNA-miRNA* duplexes from precursor miRNAs (pre-miRNAs), whereas Dicer-2 processes long double-stranded RNAs into small interfering RNA duplexes. It was recently demonstrated that Dicer-2 is rendered highly specific for long double-stranded RNA substrates by inorganic phosphate and a partner protein R2D2. However, it remains unclear how Dicer-1 exclusively recognize pre-miRNAs. Here we show that fly Dicer-1 recognizes the single-stranded terminal loop structure of pre-miRNAs through its N-terminal helicase domain, checks the loop size and measures the distance between the 3′ overhang and the terminal loop. This unique mechanism allows fly Dicer-1 to strictly inspect the authenticity of pre-miRNA structures. View full text Figures at a glance * Figure 1: Dcr-1 recognizes the terminal single-stranded region of pre-miRNAs. () Structural characteristics of typical pre-miRNAs. p*, 32P-radiolabeled phosphate. () Dcr-1 does not discriminate between internal mismatches (highlighted in orange) in the stem region. () Dcr-1 requires the loop region to be a proper size (highlighted in light blue). () Dcr-1 recognizes the terminal single-stranded region, rather than the loop structure per se. () A 7-nt single-stranded RNA on one side is not sufficient for recognition by Dcr-1. () Dcr-1 does not necessarily require single-stranded regions on both sides of the two stem strands, but rather simply requires single-stranded RNAs of sufficient length. * Figure 2: Dcr-1 measures the distance from the 3′ overhang to the terminal single-stranded region of pre-miRNAs. () Dcr-1 requires the 3′ overhang (highlighted in green) regardless of the base-pairing status at the 5′ end of pre-miRNAs. () Dcr-1 requires a stem region of the proper length (highlighted in yellow). () Dcr-1 recognizes the single-stranded RNA (highlighted in light blue) located at a proper distance from the 3′ overhang, but not necessarily at the opposite terminus from the 3′ overhang. () Dcr-1 efficiently dices wild-type pre-miR-8 but not its small-loop and long-stem variants. * Figure 3: Dcr-1 recognizes the terminal single-stranded region of pre-miRNAs through its helicase domain. () Comparison of domain structures of human DICER1 and fly Dcr-1 and Dcr-2. The poorly conserved DEXDc domain in fly Dcr-1 is shown in a faded green. The two linker insertions specific for fly Dcr-1 are shown as thick lines: L1 (371–491) and L2 (617–761). () Domain structures of the Dcr-1 deletion mutants used in –. (–) Dicing efficiency for pre-let-7 and its structural derivatives by wild-type (), ΔLinker (), ΔHelicase (), ΔDEXDc () and ΔHELICc () Dcr-1 proteins. * Figure 4: A proposed model for recognition of authentic pre-miRNA structures by Dcr-1. Dcr-1 checks the loop size by means of its helicase domain and measures the distance from the 3′ overhang to the single-stranded terminal loop region through its PAZ and helicase domains. Only when this distance is 'correct', can Dcr-1 dice the substrate through RNase IIIa and RNase IIIb domains located at a fixed distance from the 3′ overhang. Author information * Abstract * Author information * Supplementary information Affiliations * Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. * Akihisa Tsutsumi, * Tomoko Kawamata, * Natsuko Izumi & * Yukihide Tomari * Department of Medical Genome Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. * Akihisa Tsutsumi & * Yukihide Tomari * Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Moléculaire Eucaryote, Toulouse, France. * Hervé Seitz * Centre National de la Recherche Scientifique, Laboratoire de Biologie Moléculaire Eucaryote, Toulouse, France. * Hervé Seitz Contributions A.T. conducted biochemical experiments with the assistance of T.K. and N.I., H.S. conducted bioinformatic analyses, Y.T. supervised the study; A.T. and Y.T. wrote the manuscript, and all authors discussed the results and approved the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Yukihide Tomari Author Details * Akihisa Tsutsumi Search for this author in: * NPG journals * PubMed * Google Scholar * Tomoko Kawamata Search for this author in: * NPG journals * PubMed * Google Scholar * Natsuko Izumi Search for this author in: * NPG journals * PubMed * Google Scholar * Hervé Seitz Search for this author in: * NPG journals * PubMed * Google Scholar * Yukihide Tomari Contact Yukihide Tomari Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7 and Supplementary Table 1 Additional data Entities in this article * Endoribonuclease Dcr-1 Dcr-1 Drosophila melanogaster * View in UniProt * View in Entrez Gene * Dicer-2 Dcr-2 Drosophila melanogaster * View in UniProt * View in Entrez Gene * R2D2 r2d2 Drosophila melanogaster * View in UniProt * View in Entrez Gene * Drosha drosha Drosophila melanogaster * View in UniProt * View in Entrez Gene * Partner of drosha pasha Drosophila melanogaster * View in UniProt * View in Entrez Gene * Microprocessor complex subunit DGCR8 DGCR8 Homo sapiens * View in UniProt * View in Entrez Gene * Ranbp21 Ranbp21 Drosophila melanogaster * View in UniProt * View in Entrez Gene * RISC-loading complex subunit TARBP2 TARBP2 Homo sapiens * View in UniProt * View in Entrez Gene * Interferon-inducible double stranded RNA-dependent protein kinase activator A PRKRA Homo sapiens * View in UniProt * View in Entrez Gene * Protein argonaute-2 AGO2 Drosophila melanogaster * View in UniProt * View in Entrez Gene * Endoribonuclease Dicer DICER1 Homo sapiens * View in UniProt * View in Entrez Gene * let-7 let-7 Drosophila melanogaster * View in Entrez Gene * miR-8 mir-8 Drosophila melanogaster * View in Entrez Gene * Loquacious, isoform B loqs Drosophila melanogaster * View in UniProt * View in Entrez Gene * Endoribonuclease Dicer-like GL50803_103887 Giardia intestinalis (strain ATCC 50803 / WB clone C6) * View in UniProt * View in Entrez Gene * Endoribonuclease dcr-1 dcr-1 Caenorhabditis elegans * View in UniProt * View in Entrez Gene * Endoribonuclease Dicer homolog 1 DCL1 Arabidopsis thaliana * View in UniProt * View in Entrez Gene * Probable ATP-dependent RNA helicase DDX58 DDX58 Homo sapiens * View in UniProt * View in Entrez Gene - Protonation of key acidic residues is critical for the K+-selectivity of the Na/K pump
- Nat Struct Mol Biol 18(10):1159-1163 (2011)
Nature Structural & Molecular Biology | Article Protonation of key acidic residues is critical for the K+-selectivity of the Na/K pump * Haibo Yu1, 2 * Ian M Ratheal3 * Pablo Artigas3 * Benoît Roux1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1159–1163Year published:(2011)DOI:doi:10.1038/nsmb.2113Received12 March 2011Accepted04 July 2011Published online11 September 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The sodium-potassium (Na/K) pump is a P-type ATPase that generates Na+ and K+ concentration gradients across the cell membrane. For each hydrolyzed ATP molecule, the pump extrudes three Na+ and imports two K+ by alternating between outward- and inward-facing conformations that preferentially bind K+ or Na+, respectively. Remarkably, the selective K+ and Na+ binding sites share several residues, and how the pump is able to achieve the selectivity required for the functional cycle is unclear. Here, free energy–perturbation molecular dynamics (FEP/MD) simulations based on the crystal structures of the Na/K pump in a K+-loaded state (E2·Pi) reveal that protonation of the high-field acidic side chains involved in the binding sites is crucial to achieving the proper K+ selectivity. This prediction is tested with electrophysiological experiments showing that the selectivity of the E2P state for K+ over Na+ is affected by extracellular pH. View full text Figures at a glance * Figure 1: Superposition of instantaneous configurations from MD simulations of the Na/K pump. The configurations were taken at 5, 8, 11, 14, 17 and 20 ns from simulation B in Table 1. Protonation states of the binding site residues were assigned according to the theoretical prediction (thin lines), which is superimposed on the PDB 2ZXE crystal structure (thick lines). The average heavy atom r.m.s. deviations are 0.5 Å for Glu334, 0.4 Å for Glu786, 0.8 Å for Asp811 and 0.8 Å for Asp815. * Figure 2: Electrophysiological experiments on the Na/K pump expressed in Xenopus oocytes. () K+-induced mediated outward pump currents from a single Na+-loaded oocyte (Online Methods) held at −50 mV in the presence of 125 mM Na+, at two different external pHs (top, 7.6; bottom, 9.6). Vertical deflections of the current trace represent 50-ms voltage pulses (in a compressed timescale) used to obtain the half–maximally activating concentration of K+ (from Hill fits to the K+ concentration dependence of outward current). Application of 10 mM ouabain inhibits the K+-induced outward Na/K pump current. () Voltage dependence of K0.5 for K+ activation of outward pump currents at two different pHs, in the presence and absence of Na+ in the external solution. K0.5 was measured at pH 7.6 or pH 9.6, in the presence of 125 mM external Na+ (red) or 125 mM NMG. The voltage range chosen avoids the possibility that noncompetitive voltage-dependent binding of external Na+ to the Na+-exclusive site III would influence the apparent affinity for external K+. Data points represent ! mean and s.d. from five oocytes, for which titration at both pHs was evaluated. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 2ZXE * 3B8E * 1WPG * 2ZXE * 3B8E * 1WPG Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA. * Haibo Yu & * Benoît Roux * School of Chemistry, University of Wollongong, Wollongong, Australia. * Haibo Yu * Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, Texas, USA. * Ian M Ratheal & * Pablo Artigas Contributions H.Y. and B.R. designed the computations, and H.Y. carried out the computations; P.A. and B.R. set the overall aim of the experiments; the electrophysiological experiments were designed by P.A. and carried out by I.R. and P.A.; H.Y., P.A. and B.R. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Benoît Roux Author Details * Haibo Yu Search for this author in: * NPG journals * PubMed * Google Scholar * Ian M Ratheal Search for this author in: * NPG journals * PubMed * Google Scholar * Pablo Artigas Search for this author in: * NPG journals * PubMed * Google Scholar * Benoît Roux Contact Benoît Roux Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figs 1-9, Supplementary Tables 1-3 and Supplementary Methods Additional data - The export factor Yra1 modulates mRNA 3′ end processing
- Nat Struct Mol Biol 18(10):1164-1171 (2011)
Nature Structural & Molecular Biology | Article The export factor Yra1 modulates mRNA 3′ end processing * Sara A Johnson1 * Hyunmin Kim1 * Benjamin Erickson1 * David L Bentley1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1164–1171Year published:(2011)DOI:doi:10.1038/nsmb.2126Received13 March 2011Accepted20 July 2011Published online25 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The Saccharomyces cerevisiae mRNA export adaptor Yra1 binds the Pcf11 subunit of cleavage-polyadenylation factor CF1A that links export to 3′ end formation. We found that an unexpected consequence of this interaction is that Yra1 influences cleavage-polyadenylation. Yra1 competes with the CF1A subunit Clp1 for binding to Pcf11, and excess Yra1 inhibits 3′ processing in vitro. Release of Yra1 at the 3′ ends of genes coincides with recruitment of Clp1, and depletion of Yra1 enhances Clp1 recruitment within some genes. These results suggest that CF1A is not necessarily recruited as a complete unit; instead, Clp1 can be incorporated co-transcriptionally in a process regulated by Yra1. Yra1 depletion causes widespread changes in poly(A) site choice, particularly at sites where the efficiency element is divergently positioned. We propose that one way Yra1 modulates cleavage-polyadenylation is by influencing co-transcriptional assembly of the CF1A 3′ processing factor. View full text Figures at a glance * Figure 1: Yra1 influences the cleavage-polyadenylation apparatus. () Clp1 disrupts the Pcf11-Yra1 interaction. Full-length (F.L.) GST-Yra1 beads bound to Pcf11(ZCZ-His6) were incubated with RNAse-treated Clp1 or buffer (lane 3). Top panel depicts Pcf11(ZCZ-His6) that was released into the supernatant and immunoblotted. Coomassie-stained GST-Yra1 is a loading control (bottom panel). Anti-His, anti-polyhistidine () Sub2 and RNA disrupt the Pcf11-Yra1 interaction. Immunoblot of released Pcf11(ZCZ-His6), as in panel . () Yra1 inhibits coupled cleavage-polyadenylation of 32P-labeled GAL7 RNA precursors (lane 4) (see Methods). GST-Yra1 F.L. or GST-Yra1 RRM was added in lanes 2 and 3. Shown are products that were quantified by Phosphorimager and calculated ratios of polyadenylated products to precursor. Values are mean ± s.e.m (three trials). () Yra1 inhibits uncoupled cleavage and polyadenylation. Left, processing of GAL7 + cordycepin to inhibit polyadenylation. Right, polyadenylation of previously cleaved GAL7 + ATP. * Figure 2: Distinct ChIP profiles for export and 3′ processing factors. () Yra1 occupancy decreases as Clp1 occupancy increases at the 3′ ends of genes. Average distributions of Pcf11, Yra1, Clp1 and Sub2 normalized to the values at the 3′ end of the ORF on 789 protein-coding genes (Supplementary Data 1). Plot includes the ORF divided into ten equal intervals and 1 kb of 5′ and 3′ flanking sequence. Variation between these proteins in relative signals upstream of the ORF after normalization results from differences in average signal-to-background ratios between the immuniprecipitations. Note distinct transitions of export (solid arrow) and 3′ processing factors (dashed arrow) at 3′ ends. Log2 ChIP signals relative to input DNA are shown for ORFs (dotted lines, red arrow) with 1 kb of 5′ and 3′ flanking sequence (smooth lines). () Representative ChIP profiles of Pcf11, Yra1 and Clp1 generated with ChIPViewer43. Each point is the average ChIP signal at 20-base intervals. Note distinct profiles of Pcf11 and Clp1 recruitment. () Aver! age distributions as in , showing enrichment of Sub2, Yra1 and Clp1 on noncoding RNA genes, as described43. * Figure 3: Yra1 depletion enhances Clp1 recruitment on long genes. () Depletion of Yra1 in glucose grown GAL1-Yra1 cells (DBY1276-2). Immunoblots of Yra1 and Pgk1 loading control are shown. () Average ChIP-Chip profiles as in Figure 2a of Pcf11 and Clp1, normalized to the values at the 5′ end of the ORF on 128 long genes (>2,000 bases, Supplementary Data 1) in isogenic wild-type and Yra1-depleted cells (8 h in glucose). () Representative ChIP-Chip profiles of Pcf11 and Clp1 in wild-type (top row) and Yra1-depleted (bottom row) cells. Note enhanced Clp1 occupancy relative to Pcf11 within the ORFs in Yra1-depleted cells. * Figure 4: Yra1 depletion affects alternative poly(A) site choice at ACT1. () RNAse protection assay of total RNA from wild-type and Yra1-depleted GAL1-YRA1 cells (8 h glucose as in Fig. 3a). A map of the major poly(A) sites is shown below. () Reduced ORF distal and enhanced proximal site usage in Yra1-depleted cells. Cleavage at each site is expressed as a fraction of total signal after normalization for the 32P-U content of protection products 1–5. Values are mean ± s.e.m. () 3′ RNA-seq (see Methods) confirms the shift in favor of ORF proximal ACT1 poly(A) sites in isogenic wild-type and Yra1-depleted (Yra1-dep) cells (8 h in glucose, see Fig. 3a) and in the Helicos dataset41. (−) refers to transcripts on the Crick strand. The Helicos track combines reads on both strands. * Figure 5: Widespread effects of Yra1 depletion on alternative poly(A) site choice within 3′ UTRs. (,) Genome browser views of RNA-seq reads across poly(A) sites in wild-type and Yra1-depleted GAL1-YRA1 cells (8 h in glucose) and in the Helicos dataset41. (+) and (−) refer to transcripts of the Watson and Crick strands respectively. ORF proximal and ORF distal shifting of poly(A) site choice in Yra1-depleted conditions are shown in () and () respectively. Total read counts are 2.70 million for wild-type and 1.58 million for Yra1-depleted datasets. * Figure 6: Yra1 depletion alters use of poly(A) sites within genes and at ncRNAs. (–) Genome browser views of RNA-seq reads across poly(A) sites in wild-type and Yra1-depleted cells as in Figure 5. (,) Premature poly(A) sites within ORFs that are reduced () or enhanced () by Yra1 depletion. (–) Altered poly(A) site use at ncRNAs following Yra1 depletion at SUTs and CUTs (), snoRNAs () and regulatory ncRNAs (). * Figure 7: Positioning of the consensus efficiency element differs between Yra1-sensitive and Yra1-insensitive poly(A) sites. () Distribution of positioning and efficiency element (PE and EE) consensus sites relative to cleavage sites (black arrowhead, position 0) used in wild-type cells on genes that are unaffected by Yra1 depletion (nonshifters, 2,782 genes). When multiple elements were present, we chose the closest to the cleavage site. Map (right) shows location of poly(A) site consensus elements, including U-rich sequences (Ur). Red and green arrowheads mark poly(A) sites shifted upon Yra1-depletion. () Distributions of efficiency element motifs relative to cleavage sites used in wild-type cells at genes whose major poly(A) sites shift ≥15 bases following Yra1 depletion. Note the divergent positions of efficiency elements at genes that change their poly(A) site choice proximally to the ORF (349 genes, red line) or distally (381 genes, green line). () Distributions of efficiency element motifs relative to cleavage sites used in Yra1-depleted cells at the same genes shown in . Note the change ! in the position of efficiency elements (arrows) relative to cleavage sites from the wild-type cells depicted in . () A regulated assembly model for how the export factor Yra1 could influence co-transcriptional 3′ end processing. Left and right panels depict recruitment of Yra1 and CF1A subunits within genes and at 3′ ends. Rna14 and Rna15 are dotted at left to indicate that the timing of their recruitment relative to Pcf11 is not known. Poly(A) site cleavage (scissors, right panel) becomes possible at 3′ ends only after assembly of the complete CF1A complex. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE30706 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado, USA. * Sara A Johnson, * Hyunmin Kim, * Benjamin Erickson & * David L Bentley Contributions S.A.J., B.E. and D.L.B. designed and conducted the experiments. H.K. wrote the software and carried out the informatics. S.A.J., H.K. and D.L.B. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David L Bentley Author Details * Sara A Johnson Search for this author in: * NPG journals * PubMed * Google Scholar * Hyunmin Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Benjamin Erickson Search for this author in: * NPG journals * PubMed * Google Scholar * David L Bentley Contact David L Bentley Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (7M) Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Methods Excel files * Supplementary Data 1 (160K) Gene lists used in this study Additional data Entities in this article * RNA annealing protein YRA1 YRA1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Protein PCF11 PCF11 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Protein CLP1 CLP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * THO complex subunit 4 THOC4 Homo sapiens * View in UniProt * View in Entrez Gene * ATP-dependent RNA helicase SUB2 SUB2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Spliceosome RNA helicase DDX39B DDX39B Homo sapiens * View in UniProt * View in Entrez Gene * mRNA export factor MEX67 MEX67 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Nuclear RNA export factor 1 NXF1 Homo sapiens * View in UniProt * View in Entrez Gene * Nuclear polyadenylated RNA-binding protein 4 HRP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * mRNA 3'-end-processing protein RNA14 RNA14 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * mRNA 3'-end-processing protein RNA15 RNA15 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Galactose-1-phosphate uridylyltransferase GAL7 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Galactokinase GAL1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Actin ACT1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Heat shock protein SSC1, mitochondrial SSC1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Plasma membrane ATPase 1 PMA1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Reduced viability upon starvation protein 167 RVS167 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Autophagy-related protein 17 ATG17 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * ATP-dependent RNA helicase DBP2 DBP2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * ATP-dependent molecular chaperone HSP82 HSP82 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Bifunctional protein GAL10 GAL10 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Enhancer of polycomb-like protein 1 EPL1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * V-type proton ATPase subunit c' VMA11 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Flocculation protein FLO9 FLO9 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * ncRNA snR11 snR11 Saccharomyces cerevisiae S288c * View in Entrez Gene * ncRNA snR33 snR33 Saccharomyces cerevisiae S288c * View in Entrez Gene * Protein NRD1 NRD1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * ncRNA SRG1 SRG1 Saccharomyces cerevisiae S288c * View in Entrez Gene * D-3-phosphoglycerate dehydrogenase 1 SER3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Nucleolar protein 3 NPL3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Pre-mRNA cleavage complex 2 protein Pcf11 PCF11 Homo sapiens * View in UniProt * View in Entrez Gene * Polyribonucleotide 5'-hydroxyl-kinase Clp1 CLP1 Homo sapiens * View in UniProt * View in Entrez Gene * Cleavage stimulation factor subunit 2 CSTF2 Homo sapiens * View in UniProt * View in Entrez Gene * Phosphoglycerate kinase PGK1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene - Apo and InsP3-bound crystal structures of the ligand-binding domain of an InsP3 receptor
- Nat Struct Mol Biol 18(10):1172-1174 (2011)
Nature Structural & Molecular Biology | Brief Communication Apo and InsP3-bound crystal structures of the ligand-binding domain of an InsP3 receptor * Chun-Chi Lin1, 2 * Kyuwon Baek1, 2 * Zhe Lu1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1172–1174Year published:(2011)DOI:doi:10.1038/nsmb.2112Received05 April 2011Accepted01 July 2011Published online04 September 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We report the crystal structures of the ligand-binding domain (LBD) of a rat inositol 1,4,5-trisphosphate receptor (InsP3R) in its apo and InsP3-bound conformations. Comparison of these two conformations reveals that LBD's first β-trefoil fold (β-TF1) and armadillo repeat fold (ARF) move together as a unit relative to its second β-trefoil fold (β-TF2). Whereas apo LBD may spontaneously transition between gating conformations, InsP3 binding shifts this equilibrium toward the active state. View full text Figures at a glance * Figure 1: Structures of LBD. (,) Surface and ribbon representations of LBD structures without () and with () InsP3 bound, with β-TF2 shown in the same orientation. Surfaces of β-TF1, β-TF2, ARF and InsP3 are colored light blue, yellow, pink and orange, respectively; α helices, β-strands, linkers between lobes, and the InsP3 molecule, are colored lime, magenta, blue and orange, respectively. (,) View of LBD structures rotated 90° from and , where surfaces of the three lobes are colored as in and , and ribbon representations of β-TF1, β-TF2 and ARF are colored blue, orange and magenta, respectively. InsP3 is shown in lime sticks. Solid and dotted lines indicate the axes of helix α4 in the bound and unbound states, respectively. * Figure 2: Structures and electron density maps of regions within LDB. () Stereo view of a section (Asp442–Leu453) of helix α4 in the InsP3-bound structure, superimposed on the corresponding 2Fo – Fc map contoured at 1 σ. (,) Structures of the InsP3-binding site in LBD bound () or unbound () with InsP3, superimposed on the respective Fo – Fc InsP3 omit maps contoured at 4 σ. The InsP3 molecule in panel is shown as a stick model; the corresponding site in is delineated by dots. The InsP3-interacting side chains, shown as sticks in , are mostly disordered in . The red dotted lines in indicate potential hydrogen bonds. * Figure 3: Comparison of InsP3-bound and InsP3-unbound LBD structures. (,) ARF () and β-TF1 () structures of bound (blue) and unbound (yellow) LBD are aligned using β-TF2 as a reference. (,) ARF () and β-TF2 () structures of bound (blue) and unbound (yellow) LBD are aligned using β-TF1 as a reference. () Shown are ARF structures of InsP3-bound (blue) and InsP3-unbound (yellow) LBD as well as that of the InsP3-bound partial LBD (magenta) (PDB 1N4K), all aligned using β-TF2 as reference. For clarity, β-TF1 or β-TF2 or both are removed, and for easy comparison, the C-terminal 581–602 region of the partial LBD (PDB 1N4K) is not shown, as it is disordered in both LBD structures. Accession codes * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3T8S * 3T8S Referenced accessions Protein Data Bank * 1XZZ * 1N4K * 1XZZ * 1N4K Author information * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Chun-Chi Lin & * Kyuwon Baek Affiliations * Department of Physiology, Howard Hughes Medical Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Chun-Chi Lin, * Kyuwon Baek & * Zhe Lu Contributions C.-C.L., K.B. and Z.L. conducted the experiments, analyzed the data and prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Zhe Lu Author Details * Chun-Chi Lin Search for this author in: * NPG journals * PubMed * Google Scholar * Kyuwon Baek Search for this author in: * NPG journals * PubMed * Google Scholar * Zhe Lu Contact Zhe Lu Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (15M) Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods Movies * Supplementary Movie 1 (2M) LBD structures with and without InsP3 bound, viewed alternatingly as in Fig. 1a,b, then as in Fig. 1c,d, followed by a zoom-in view of the InsP3-binding region with InsP3-interacting side chains. β-TF2 was used as reference to align the bound and unbound structures. Additional data Entities in this article * Inositol 1,4,5-trisphosphate receptor type 1 Itpr1 Rattus norvegicus * View in UniProt * View in Entrez Gene - Crystal structure of a monomeric retroviral protease solved by protein folding game players
- Nat Struct Mol Biol 18(10):1175-1177 (2011)
Nature Structural & Molecular Biology | Brief Communication Crystal structure of a monomeric retroviral protease solved by protein folding game players * Firas Khatib1 * Frank DiMaio1 * Foldit Contenders Group * Foldit Void Crushers Group * Seth Cooper2 * Maciej Kazmierczyk3 * Miroslaw Gilski3, 4 * Szymon Krzywda3 * Helena Zabranska5 * Iva Pichova5 * James Thompson1 * Zoran Popović2 * Mariusz Jaskolski3, 4 * David Baker1, 6 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1175–1177Year published:(2011)DOI:doi:10.1038/nsmb.2119Received27 May 2011Accepted08 July 2011Published online18 September 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Following the failure of a wide range of attempts to solve the crystal structure of M-PMV retroviral protease by molecular replacement, we challenged players of the protein folding game Foldit to produce accurate models of the protein. Remarkably, Foldit players were able to generate models of sufficient quality for successful molecular replacement and subsequent structure determination. The refined structure provides new insights for the design of antiretroviral drugs. View full text Figures at a glance * Figure 1: Successful CASP9 predictions by the Foldit Void Crushers Group. () Starting from the fourth-ranked Rosetta Server model (red) for CASP9 target T0581, the Foldit Void Crushers Group (yellow) generated a model that was closer to the crystal structure later determined (blue). () Starting from a modified Rosetta model built using the Alignment Tool (red), the Foldit Void Crushers Group generated a model (yellow) considerably closer to the later determined crystal structure (blue). Images were produced using PyMOL software (http://www.pymol.org). * Figure 2: M-PMV retroviral protease structure improvement by the Foldit Contenders Group. () Progress of structure refinement over the first 16 d of game play. The x axis shows progression in time, and the y axis shows the Phaser log-likelihood (LLG) of each model in a near-native orientation. To identify a solution as correct by molecular replacement using Phaser, the model must have an LLG better than the best random models. The distribution of these best random predictions is indicated by the intensity of the pale blue band. (Because almost all the models are too poor to allow correct placement in the unit cell, Phaser LLGs are calculated after optimal superposition of each model onto the solved crystal structure and rigid-body optimization.) () Starting from a quite inaccurate NMR model (red), Foldit player spvincent generated a model (yellow) considerably more similar to the later determined crystal structure (blue) in the β-strand region. () Starting from spvincent's model, Foldit player grabhorn generated a model (magenta) considerably closer to the cryst! al structure with notable improvement of side-chain conformations in the hydrophobic core. () Foldit player mimi made additional improvements (in the loop region at the top left) and generated a model (green) of sufficient accuracy to provide an unambiguous molecular replacement solution which allowed rapid determination of the ultimate crystal structure (blue). * Figure 3: CPK representation of retropepsin surface. () The surface of HIV-1 PR protomer extracted from the dimeric molecule (PDB 3hvp), as seen from the direction of the removed dimerization partner. () M-PMV PR monomer shown in the same orientation and scale. In this view, the N and C termini (missing in M-PMV PR) are at the bottom, and the flap loop is at the top. The active-site cavity (ASC) is clearly seen between the flap and the body of the HIV-1 PR molecule. In M-PMV PR, the cavity is completely covered by the curled flap. Accession codes * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3SQF * 3SQF Referenced accessions Protein Data Bank * 3hvp * 3hvp Author information * Accession codes * Author information * Supplementary information Affiliations * Department of Biochemistry, University of Washington, Seattle, Washington, USA. * Firas Khatib, * Frank DiMaio, * James Thompson & * David Baker * Department of Computer Science and Engineering, University of Washington, Seattle, Washington, USA. * Seth Cooper & * Zoran Popović * Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland. * Maciej Kazmierczyk, * Miroslaw Gilski, * Szymon Krzywda & * Mariusz Jaskolski * Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland. * Miroslaw Gilski & * Mariusz Jaskolski * Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Poznan, Czech Republic. * Helena Zabranska & * Iva Pichova * Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA. * David Baker Consortia * Foldit Contenders Group * Foldit Void Crushers Group Contributions F.K., F.D., S.C., J.T., Z.P. and D.B. contributed to the development and analysis of Foldit and to the writing of the manuscript; the F.C.G. and F.V.C.G. contributed through their gameplay, which generated the results for this manuscript; M.K. grew the crystals and collected X-ray diffraction data; M.G. processed X-ray data and analyzed the structure; S.K. refined the structure; H.Z. cloned, expressed and purified the protein; I.P. designed and coordinated the biochemical experiments, and contributed to writing the manuscript; M.J. coordinated the crystallographic study, analyzed the results and contributed to writing the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David Baker Author Details * Firas Khatib Search for this author in: * NPG journals * PubMed * Google Scholar * Frank DiMaio Search for this author in: * NPG journals * PubMed * Google Scholar * Foldit Contenders Group * Foldit Void Crushers Group * Seth Cooper Search for this author in: * NPG journals * PubMed * Google Scholar * Maciej Kazmierczyk Search for this author in: * NPG journals * PubMed * Google Scholar * Miroslaw Gilski Search for this author in: * NPG journals * PubMed * Google Scholar * Szymon Krzywda Search for this author in: * NPG journals * PubMed * Google Scholar * Helena Zabranska Search for this author in: * NPG journals * PubMed * Google Scholar * Iva Pichova Search for this author in: * NPG journals * PubMed * Google Scholar * James Thompson Search for this author in: * NPG journals * PubMed * Google Scholar * Zoran Popović Search for this author in: * NPG journals * PubMed * Google Scholar * Mariusz Jaskolski Search for this author in: * NPG journals * PubMed * Google Scholar * David Baker Contact David Baker Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–3, Supplementary Table 1 and Supplementary Discussion Additional data
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