Hot off the presses! Sep 24 mol cell

The Sep 24 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:

• A Mitotic Beacon Reveals Its Nucleosome Anchor
  - mol cell 39(6):829-830 (2010)
  Mitosis, nuclear envelope formation, and nucleocytoplasmic transport require chromosomes to identify themselves by enriching Ran-GTP around the chromatin fiber. In a recent Nature report, Makde et al. (2010) describe the structure of the Ran activator RCC1 anchored onto nucleosomes.
• The End of the Circle for Yeast Mitochondrial DNA
  - mol cell 39(6):831-832 (2010)
  In this issue of Molecular Cell, Gerhold et al. (2010) find no circular DNA during mitochondrial DNA (mtDNA) replication in the aerobic yeast Candida albicans, a result with important implications for mtDNA replication in Saccharomyces cerevisiae.
• Long Noncoding RNAs Add Another Layer to Pre-mRNA Splicing Regulation
  - mol cell 39(6):833-834 (2010)
  In this issue of Molecular Cell, Tripathi and coworkers (Tripathi et al., 2010) decode some of the functions of a long noncoding RNA MALAT1. They provide evidence that MALAT1 regulates alternative splicing by controlling the activity of the SR protein family of splicing factors.
• aMAGEing New Players Enter the RING to Promote Ubiquitylation
  - mol cell 39(6):835-837 (2010)
  The MAGE proteins are best known as curious tumor-specific antigens. However, Doyle et al. (2010) reveal that MAGE proteins interact with RING proteins to promote ubiquitylation which provides important new insights into the physiological and pathological functions of this enigmatic family of proteins.
• CK2 Phospho-Dependent Binding of R2TP Complex to TEL2 Is Essential for mTOR and SMG1 Stability
  - mol cell 39(6):839-850 (2010)
  TEL2 interacts with and is essential for the stability of all phosphatidylinositol 3-kinase-related kinases (PIKKs), but its mechanism of action remains unclear. Here, we show that TEL2 is constitutively phosphorylated on conserved serines 487 and 491 by casein kinase 2 (CK2). Proteomic analyses establish that the CK2 phosphosite of TEL2 confers binding to the R2TP/prefoldin-like complex, which possesses chaperon/prefoldin activities required during protein complex assembly. The PIH1D1 subunit of the R2TP complex binds directly to the CK2 phosphosite of TEL2 in vitro and is required for the TEL2-R2TP/prefoldin-like complex interaction in vivo. Although the CK2 phosphosite mutant of TEL2 retains association with the PIKKs and HSP90 in cells, failure to interact with the R2TP/prefoldin-like complex results in instability of the PIKKs, principally mTOR and SMG1. We propose that TEL2 acts as a scaffold to coordinate the activities of R2TP/prefoldin-like and HSP90 chaperone! complexes during the assembly of the PIKKs.
• Strand Invasion Structures in the Inverted Repeat of Candida albicans Mitochondrial DNA Reveal a Role for Homologous Recombination in Replication
  - mol cell 39(6):851-861 (2010)
  Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study ! wild-type mtDNA maintenance in yeast.
• Swi2/Snf2-Related Translocases Prevent Accumulation of Toxic Rad51 Complexes during Mitotic Growth
  - mol cell 39(6):862-872 (2010)
  Purified DNA translocases Rdh54 and Rad54 can dissociate complexes formed by eukaryotic RecA-like recombinases on double-stranded DNA. Here, we show that Rad51 complexes are dissociated by these translocases in mitotic cells. Rad51 overexpression blocked growth of cells deficient in Rdh54 activity. This toxicity was associated with accumulation of Rad51 foci on undamaged chromatin. At normal Rad51 levels, rdh54 deficiency resulted in slight elevation of Rad51 foci. A triple mutant lacking Rdh54, Rad54, and a third Swi2/Snf2 homolog Uls1 accumulated Rad51 foci, grew slowly, and suffered chromosome loss. Thus, Uls1 and Rad54 can partially substitute for Rdh54 in the removal of toxic, nondamage-associated Rad51-DNA complexes. Additional data suggest that the function of Rdh54 and Rad54 in removal of Rad51 foci is significantly specialized; Rad54 predominates for removal of damage-associated foci, and Rdh54 predominates for removal of nondamage-associated foci.
• AID-Induced Genotoxic Stress Promotes B Cell Differentiation in the Germinal Center via ATM and LKB1 Signaling
  - mol cell 39(6):873-885 (2010)
  During an immune response, B cells undergo rapid proliferation and activation-induced cytidine deaminase (AID)-dependent remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, the genotoxic stress associated with the GC reaction also promotes most B cell malignancies. Here, we report that exogenous and intrinsic AID-induced DNA strand breaks activate ATM, which signals through an LKB1 intermediate to inactivate CRTC2, a transcriptional coactivator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation while opposing lymphomagenesis. Inhibition of this pathway results in increased GC B cell proliferation, reduced antibody secretion, and impaired terminal differentiation. Multiple distinct pathway disruptions were also identified in human GC B cell lymphoma patie! nt samples. Combined, our data show that CRTC2 inactivation, via physiologic DNA damage response signaling, promotes B cell differentiation in response to genotoxic stress.
• Polycomb Group Protein Displacement and Gene Activation through MSK-Dependent H3K27me3S28 Phosphorylation
  - mol cell 39(6):886-900 (2010)
  Epigenetic regulation of chromatin structure is essential for the expression of genes determining cellular specification and function. The Polycomb repressive complex 2 (PRC2) di- and trimethylates histone H3 on lysine 27 (H3K27me2/me3) to establish repression of specific genes in embryonic stem cells and during differentiation. How the Polycomb group (PcG) target genes are regulated by environmental cues and signaling pathways is quite unexplored. Here, we show that the mitogen- and stress-activated kinases (MSK), through a mechanism that involves promoter recruitment, histone H3K27me3S28 phosphorylation, and displacement of PcG proteins, lead to gene activation. We present evidence that the H3K27me3S28 phosphorylation is functioning in response to stress signaling, mitogenic signaling, and retinoic acid (RA)-induced neuronal differentiation. We propose that MSK-mediated H3K27me3S28 phosphorylation serves as a mechanism to activate a subset of PcG target genes determi! ned by the biological stimuli and thereby modulate the gene expression program determining cell fate.
• H2A.Z Maintenance during Mitosis Reveals Nucleosome Shifting on Mitotically Silenced Genes
  - mol cell 39(6):901-911 (2010)
  Profound chromatin changes occur during mitosis to allow for gene silencing and chromosome segregation followed by reactivation of memorized transcription states in daughter cells. Using genome-wide sequencing, we found H2A.Z-containing +1 nucleosomes of active genes shift upstream to occupy TSSs during mitosis, significantly reducing nucleosome-depleted regions. Single-molecule analysis confirmed nucleosome shifting and demonstrated that mitotic shifting is specific to active genes that are silenced during mitosis and, thus, is not seen on promoters, which are silenced by methylation or mitotically expressed genes. Using the GRP78 promoter as a model, we found H3K4 trimethylation is also maintained while other indicators of active chromatin are lost and expression is decreased. These key changes provide a potential mechanism for rapid silencing and reactivation of genes during the cell cycle.
• HSP90 and Its R2TP/Prefoldin-like Cochaperone Are Involved in the Cytoplasmic Assembly of RNA Polymerase II
  - mol cell 39(6):912-924 (2010)
  RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, su! ggesting a general role in assembling RNA polymerases.
• The Nuclear-Retained Noncoding RNA MALAT1 Regulates Alternative Splicing by Modulating SR Splicing Factor Phosphorylation
  - mol cell 39(6):925-938 (2010)
  Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the r! ole for an nrRNA in the regulation of gene expression.
• Structural Basis for Substrate Placement by an Archaeal Box C/D Ribonucleoprotein Particle
  - mol cell 39(6):939-949 (2010)
  Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2′-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a half-mer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modeling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide ! RNA and large ribosomal and spliceosomal substrate RNAs.
• The 5′-7-Methylguanosine Cap on Eukaryotic mRNAs Serves Both to Stimulate Canonical Translation Initiation and to Block an Alternative Pathway
  - mol cell 39(6):950-962 (2010)
  Translational control is frequently exerted at the stage of mRNA recruitment to the initiating ribosome. We have reconstituted mRNA recruitment to the 43S preinitiation complex (PIC) using purified S. cerevisiae components. We show that eIF3 and the eIF4 factors not only stabilize binding of mRNA to the PIC, they also dramatically increase the rate of recruitment. Although capped mRNAs require eIF3 and the eIF4 factors for efficient recruitment to the PIC, uncapped mRNAs can be recruited in the presence of eIF3 alone. The cap strongly inhibits this alternative recruitment pathway, imposing a requirement for the eIF4 factors for rapid and stable binding of natural mRNA. Our data suggest that the 5′ cap serves as both a positive and negative element in mRNA recruitment, promoting initiation in the presence of the canonical group of mRNA handling factors while preventing binding to the ribosome via an aberrant, alternative pathway requiring only eIF3.
• MAGE-RING Protein Complexes Comprise a Family of E3 Ubiquitin Ligases
  - mol cell 39(6):963-974 (2010)
  The melanoma antigen (MAGE) family consists of more than 60 genes, many of which are cancer-testis antigens that are highly expressed in cancer and play a critical role in tumorigenesis. However, the biochemical and cellular functions of this enigmatic family of proteins have remained elusive. Here, we identify really interesting new gene (RING) domain proteins as binding partners for MAGE family proteins. Multiple MAGE family proteins bind E3 RING ubiquitin ligases with specificity. The crystal structure of one of these MAGE-RING complexes, MAGE-G1-NSE1, reveals structural insights into MAGE family proteins and their interaction with E3 RING ubiquitin ligases. Biochemical and cellular assays demonstrate that MAGE proteins enhance the ubiquitin ligase activity of RING domain proteins. For example, MAGE-C2-TRIM28 is shown to target p53 for degradation in a proteasome-dependent manner, consistent with its tumorigenic functions. These findings define a biochemical and cel! lular function for the MAGE protein family.
• Imaging-Based Identification of a Critical Regulator of FtsZ Protofilament Curvature in Caulobacter
  - mol cell 39(6):975-987 (2010)
  FtsZ is an essential bacterial GTPase that polymerizes at midcell, recruits the division machinery, and may generate constrictive forces necessary for cytokinesis. However, many of the mechanistic details underlying these functions are unknown. We sought to identify FtsZ-binding proteins that influence FtsZ function in Caulobacter crescentus. Here, we present a microscopy-based screen through which we discovered two FtsZ-binding proteins, FzlA and FzlC. FzlA is conserved in α-proteobacteria and was found to be functionally critical for cell division in Caulobacter. FzlA altered FtsZ structure both in vivo and in vitro, forming stable higher-order structures that were resistant to depolymerization by MipZ, a spatial determinant of FtsZ assembly. Electron microscopy revealed that FzlA organizes FtsZ protofilaments into striking helical bundles. The degree of curvature induced by FzlA depended on the nucleotide bound to FtsZ. Induction of FtsZ curvature by FzlA carries i! mplications for regulating FtsZ function by modulating its superstructure.