Hot off the presses! May 15 Mol Cell

The May 15 issue of the Mol Cell is now up on Pubget (About Mol Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Of Elections and Cell-Death Decisions
  - Mol Cell 34(3):257-258 (2009)
  A new study in the journal Nature (Spencer et al., 2009) argues that cell-to-cell variation in the decision to undergo apoptosis is not due to genetic, epigenetic, or cell-cycle differences, nor due to random molecular noise, but instead is determined by differences in protein abundances.
• A Role for Ubiquitin in Selective Autophagy
  - Mol Cell 34(3):259-269 (2009)
  Ubiquitination is the hallmark of protein degradation by the 26S proteasome. However, the proteasome is limited in its capacity to degrade oligomeric and aggregated proteins. Removal of harmful protein aggregates is mediated by autophagy, a mechanism by which the cell sequesters cytosolic cargo and delivers it for degradation by the lysosome. Identification of autophagy receptors, such as p62/SQSTM1 and NBR1, which simultaneously bind both ubiquitin and autophagy-specific ubiquitin-like modifiers, LC3/GABARAP, has provided a molecular link between ubiquitination and autophagy. This review explores the hypothesis that ubiquitin represents a selective degradation signal suitable for targeting various types of cargo, ranging from protein aggregates to membrane-bound organelles and microbes.
• Epigenetic Silencing of the p16INK4a Tumor Suppressor Is Associated with Loss of CTCF Binding and a Chromatin Boundary
  - Mol Cell 34(3):271-284 (2009)
  The p16INK4a tumor suppressor gene is a frequent target of epigenetic inactivation in human cancers, which is an early event in breast carcinogenesis. We describe the existence of a chromatin boundary upstream of the p16 gene that is lost when this gene is aberrantly silenced. We show that the multifunctional protein CTCF associates in the vicinity of this boundary and absence of binding strongly coincides with p16 silencing in multiple types of cancer cells. CTCF binding also correlates with RASSF1A and CDH1 gene activation, and CTCF interaction is absent when these genes are methylated and silenced. Interestingly, defective poly(ADP-ribosyl)ation of CTCF and dissociation from the molecular chaperone Nucleolin occur in p16-silenced cells, abrogating its proper function. Thus, destabilization of specific chromosomal boundaries through aberrant crosstalk between CTCF, poly(ADP-ribosyl)ation, and DNA methylation may be a general mechanism to inactivate tumor suppressor g! enes and initiate tumorigenesis in numerous forms of human cancers.
• Essential Role for DNA-PKcs in DNA Double-Strand Break Repair and Apoptosis in ATM-Deficient Lymphocytes
  - Mol Cell 34(3):285-297 (2009)
  The DNA double-strand break (DSB) repair protein DNA-PKcs and the signal transducer ATM are both activated by DNA breaks and phosphorylate similar substrates in vitro, yet appear to have distinct functions in vivo. Here, we show that ATM and DNA-PKcs have overlapping functions in lymphocytes. Ablation of both kinase activities in cells undergoing immunoglobulin class switch recombination leads to a compound defect in switching and a synergistic increase in chromosomal fragmentation, DNA insertions, and translocations due to aberrant processing of DSBs. These abnormalities are attributed to a compound deficiency in phosphorylation of key proteins required for DNA repair, class switching, and cell death. Notably, both kinases are required for normal levels of p53 phosphorylation in B and T cells and p53-dependent apoptosis. Our experiments reveal a DNA-PKcs-dependent pathway that regulates DNA repair and activation of p53 in the absence of ATM.
• Formation of Dynamic γ-H2AX Domains along Broken DNA Strands Is Distinctly Regulated by ATM and MDC1 and Dependent upon H2AX Densities in Chromatin
  - Mol Cell 34(3):298-310 (2009)
  A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate γ-H2AX. Here, we demonstrate that γ-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which γ-H2AX forms does not spread as DSBs persist; rather, γ-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate γ-H2AX, only ATM promotes γ-H2AX formation to maximal distance and maintains γ-H2AX densities. MDC1 is essential for γ-H2AX formation at high densities near DSBs, but not for generation of γ-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of γ-H2AX formed. Our data suggest that H2AX fuels a γ-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX.
• Distinct Targets of the Eco1 Acetyltransferase Modulate Cohesion in S Phase and in Response to DNA Damage
  - Mol Cell 34(3):311-321 (2009)
  Chromosome segregation and the repair of DNA double-strand breaks (DSBs) require cohesin, the protein complex that mediates sister chromatid cohesion. Cohesion requires both a chromatin binding step and a subsequent tethering step called cohesion generation. Here we provide insight into how cohesion generation is restricted to S phase but can be activated in G2/M by a DSB in budding yeast. We show that Wpl1p inhibits cohesion in G2/M. A DSB counteracts Wpl1p and stimulates cohesion generation by first inducing the phosphorylation of the Mcd1p subunit of cohesin. This phosphorylation activates Eco1p-dependent acetylation of Mcd1p, which in turn antagonizes Wpl1p. Previous studies show that Eco1p antagonizes Wpl1p in S phase by acetylating the Smc3p subunit of cohesin. We show that Mcd1p and Smc3p acetylation antagonize Wpl1p only in their proper context. Thus, Eco1p antagonizes Wpl1p in distinct ways to modulate cohesion generation during the cell cycle and after DNA da! mage.
• A Core Complex of CPSF73, CPSF100, and Symplekin May Form Two Different Cleavage Factors for Processing of Poly(A) and Histone mRNAs
  - Mol Cell 34(3):322-332 (2009)
  Metazoan histone mRNAs are unique: their pre-mRNAs contain no introns, and the mRNAs are not polyadenylated, ending instead in a conserved stem-loop structure. In Drosophila, canonical poly(A) signals are located downstream of the normal cleavage site of each histone gene and are utilized when histone 3′ end formation is inhibited. Here we define a subcomplex of poly(A) factors that are required for histone pre-mRNA processing. We demonstrate that Symplekin, CPSF73, and CPSF100 are present in a stable complex and interact with histone-specific processing factors. We use chromatin immunoprecipitation to show that Symplekin and CPSF73, but not CstF50, cotranscriptionally associate with histone genes. Depletion of SLBP recruits CstF50 to histone genes. Knockdown of CPSF160 or CstF64 downregulates Symplekin but does not affect histone pre-mRNA processing or association of Symplekin with the histone locus. These results suggest that a common core cleavage factor is requir! ed for processing of histone and polyadenylated pre-mRNAs.
• Insights into Branch Nucleophile Positioning and Activation from an Orthogonal Pre-mRNA Splicing System in Yeast
  - Mol Cell 34(3):333-343 (2009)
  The duplex formed between the branch site (BS) of a spliceosomal intron and its cognate sequence in U2 snRNA is important for spliceosome assembly and the first catalytic step of splicing. We describe the development of an orthogonal BS-U2 system in S. cerevisiae in which spliceosomes containing a grossly substituted second-copy U2 snRNA mediate the in vivo splicing of a single reporter transcript carrying a cognate substitution. Systematic use of this approach to investigate requirements for branching catalysis reveals considerable flexibility in the sequence of the BS-U2 duplex and its positioning relative to the catalytic center. Branching efficiency depends on the identity of the branch nucleotide, its position within the BS-U2 duplex, and its distance from U2/U6 helix Ia. These results provide insights into substrate selection during spliceosomal branching catalysis; additionally, this system provides a foundation and tool for future mechanistic splicing research.
• YidC and Oxa1 Form Dimeric Insertion Pores on the Translating Ribosome
  - Mol Cell 34(3):344-353 (2009)
  The YidC/Oxa1/Alb3 family of membrane proteins facilitates the insertion and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here we present the structures of both Escherichia coli YidC and Saccharomyces cerevisiae Oxa1 bound to E. coli ribosome nascent chain complexes determined by cryo-electron microscopy. Dimers of YidC and Oxa1 are localized above the exit of the ribosomal tunnel. Crosslinking experiments show that the ribosome specifically stabilizes the dimeric state. Functionally important and conserved transmembrane helices of YidC and Oxa1 were localized at the dimer interface by cysteine crosslinking. Both Oxa1 and YidC dimers contact the ribosome at ribosomal protein L23 and conserved rRNA helices 59 and 24, similarly to what was observed for the nonhomologous SecYEG translocon. We suggest that dimers of the YidC and Oxa1 proteins form insertion pores and share a common overall architecture with the SecY monomer.
• Quantitative Proteomics Reveals a Dynamic Interactome and Phase-Specific Phosphorylation in the Neurospora Circadian Clock
  - Mol Cell 34(3):354-363 (2009)
  Circadian systems are comprised of multiple proteins functioning together to produce feedback loops driving robust, approximately 24 hr rhythms. In all circadian systems, proteins in these loops are regulated through myriad physically and temporally distinct posttranslational modifications (PTMs). To better understand how PTMs impact a circadian oscillator, we implemented a proteomics-based approach by combining purification of endogenous FREQUENCY (FRQ) and its interacting partners with quantitative mass spectrometry (MS). We identify and quantify time-of-day-specific protein-protein interactions in the clock and show how these provide a platform for temporal and physical separation between the dual roles of FRQ. Additionally, by unambiguously identifying over 75 phosphorylated residues, following their quantitative change over a circadian cycle, and examining the phenotypes of strains that have lost these sites, we demonstrate how spatially and temporally regulated p! hosphorylation has opposing effects directly on overt circadian rhythms and FRQ stability.
• Coronin Switches Roles in Actin Disassembly Depending on the Nucleotide State of Actin
  - Mol Cell 34(3):364-374 (2009)
  Rapid and polarized turnover of actin networks is essential for motility, endocytosis, cytokinesis, and other cellular processes. However, the mechanisms that provide tight spatiotemporal control of actin disassembly remain poorly understood. Here, we show that yeast coronin (Crn1) makes a unique contribution to this process by differentially interacting with and regulating the effects of cofilin on ATP/ADP+Pi versus ADP actin filaments. Crn1 potently blocks cofilin severing of newly assembled (ATP/ADP+Pi) filaments but synergizes with cofilin to sever older (ADP) filaments. Thus, Crn1 has qualitatively distinct/opposite effects on actin dynamics depending on the nucleotide state of actin. This bimodal mechanism requires two separate actin-binding domains in Crn1. Consistent with these activities, Crn1 excludes GFP-Cof1 from newly assembled regions of actin networks in vivo and accelerates cellular actin turnover by four fold. We conclude that coronin polarizes the spa! tial distribution and activity of cofilin to promote selective disassembly of older actin filaments.
• Crystallographic Insights into the Autocatalytic Assembly Mechanism of a Bacteriophage Tail Spike
  - Mol Cell 34(3):375-386 (2009)
  The tailed bacteriophage phi29 has 12 "appendages" (gene product 12, gp12) attached to its neck region that participate in host cell recognition and entry. In the cell, monomeric gp12 undergoes proteolytic processing that releases the C-terminal domain during assembly into trimers. We report here crystal structures of the protein before and after catalytic processing and show that the C-terminal domain of gp12 is an "autochaperone" that aids trimerization. We also show that autocleavage of the C-terminal domain is a posttrimerization event that is followed by a unique ATP-dependent release. The posttranslationally modified N-terminal part has three domains that function to attach the appendages to the phage, digest the cell wall teichoic acids, and bind irreversibly to the host, respectively. Structural and sequence comparisons suggest that some eukaryotic and bacterial viruses as well as bacterial adhesins might have a similar maturation mechanism as is perfor! med by phi29 gp12 for Bacillus subtilis.
• TFIIH Kinase Places Bivalent Marks on the Carboxy-Terminal Domain of RNA Polymerase II
  - Mol Cell 34(3):387-393 (2009)
  Posttranslational modifications of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) specify a molecular recognition code that is deciphered by proteins involved in RNA biogenesis. The CTD is comprised of a repeating heptapeptide (Y1S2P3T4S5P6S7). Recently, phosphorylation of serine 7 was shown to be important for cotranscriptional processing of two snRNAs in mammalian cells. Here we report that Kin28/Cdk7, a subunit of the evolutionarily conserved TFIIH complex, is a Ser7 kinase. The ability of Kin28/Cdk7 to phosphorylate Ser7 is particularly surprising because this kinase functions at promoters of protein-coding genes, rather than being restricted to promoter-distal regions of snRNA genes. Kin28/Cdk7 is also known to phosphorylate Ser5 residues of the CTD at gene promoters. Taken together, our results implicate the TFIIH kinase in placing bivalent Ser5 and Ser7 marks early in gene transcription. These bivalent CTD marks, in concer! t with cues within nascent transcripts, specify the cotranscriptional engagement of the relevant RNA processing machinery.