Hot off the presses! Dec 22 mol cell

The Dec 22 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:

• UNCovering the Molecular Machinery of Dependence Receptor Signaling
  - mol cell 40(6):851-853 (2010)
  Dependence receptors send opposite signals in the presence or absence of ligand, but the underlying mechanisms have been elusive. In this issue of Molecular Cell, Guenebeaud et al. (2010) elucidate the molecular signaling machinery of the dependence receptor UNC5B.
• Targeted Ubiquitylation: The Prey Becomes Predator
  - mol cell 40(6):853-855 (2010)
  Being targeted for polyubiquitylation often means the end of life for the substrate protein. In this issue of Molecular Cell, Ouni et al. (2010) demonstrate that the yeast transcription factor Met4, a target of the SCFMet30 E3 ligase for nonproteolytic polyubiquitylation, can also function to target its cofactors for proteolytic ubiquitylation by the same E3 ligase.
• The Three Rs of Transcription: Recruit, Retain, and Recycle
  - mol cell 40(6):855-858 (2010)
  The dynamic protein interactions required for transcription are functionally important yet poorly understood; in this issue of Molecular Cell, Zobeck et al. (2010) resolve the sequential recruitment and selective recycling of transcription factors at an actively transcribing locus in Drosophila.
• Seeking Resolution: Budding Yeast Enzymes Finally Make the Cut
  - mol cell 40(6):858-859 (2010)
  Genetic studies reported in Molecular Cell (Ho et al., 2010) identify Mus81-Mms4 and Yen1 as the structure-specific endonucleases that cleave most Holliday junctions. A failure in this key step has profound effects on mitotic genome stability.
• DNA Replication: Making Two Forks from One Prereplication Complex
  - mol cell 40(6):860-861 (2010)
  The copying of chromosomal DNA initiates from a single nucleoprotein assembly called the prereplication complex. New findings in a recent issue of Molecular Cell (Yardimci et al., 2010) reveal that this complex dissolves into two independent replisomes that move away from each other as DNA synthesis ensues.
• The Dependence Receptor UNC5H2/B Triggers Apoptosis via PP2A-Mediated Dephosphorylation of DAP Kinase
  - mol cell 40(6):863-876 (2010)
  The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65β of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65β and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphoryla! tion and that this mechanism is involved in angiogenesis regulation.
• Identification of the Rac-GEF P-Rex1 as an Essential Mediator of ErbB Signaling in Breast Cancer
  - mol cell 40(6):877-892 (2010)
  While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here, we identify the PIP3-Gβγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gβγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector p! athway that is crucial for breast cancer progression.
• Sirt3-Mediated Deacetylation of Evolutionarily Conserved Lysine 122 Regulates MnSOD Activity in Response to Stress
  - mol cell 40(6):893-904 (2010)
  Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3−/− livers at 3 months of age. Livers of Sirt3−/− mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null Sirt3 into Sirt3−/− MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSODK122-R), increased MnSOD activity when expressed in MnSOD−/− MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3−/− MEFs with lenti-MnSODK122-R inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and ! decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3−/− livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.
• Structural Basis for Oligosaccharide Recognition of Misfolded Glycoproteins by OS-9 in ER-Associated Degradation
  - mol cell 40(6):905-916 (2010)
  Misfolded glycoproteins are translocated from endoplasmic reticulum (ER) into the cytosol for proteasome-mediated degradation. A mannose-6-phosphate receptor homology (MRH) domain is commonly identified in a variety of proteins and, in the case of OS-9 and XTP3-B, is involved in glycoprotein ER-associated degradation (ERAD). Trimming of outermost α1,2-linked mannose on C-arm of high-mannose-type glycan and binding of processed α1,6-linked mannosyl residues by the MRH domain are critical steps in guiding misfolded glycoproteins to enter ERAD. Here we report the crystal structure of a human OS-9 MRH domain (OS-9MRH) complexed with α3,α6-mannopentaose. The OS-9MRH has a flattened β-barrel structure with a characteristic P-type lectin fold and possesses distinctive double tryptophan residues in the oligosaccharide-binding site. Our crystallographic result in conjunction with nuclear magnetic resonance (NMR) spectroscopic and biochemical results provides structural ins! ights into the mechanism whereby OS-9 specifically recognizes Manα1,6Manα1,6Man residues on the processed C-arm through the continuous double tryptophan (WW) motif.
• Ubiquitylation of an ERAD Substrate Occurs on Multiple Types of Amino Acids
  - mol cell 40(6):917-926 (2010)
  Any protein synthesized in the secretory pathway has the potential to misfold and would need to be recognized and ubiquitylated for degradation. This is astounding, since only a few ERAD-specific E3 ligases have been identified. To begin to understand substrate recognition, we wished to map the ubiquitylation sites on the NS-1 nonsecreted immunoglobulin light chain, which is an ERAD substrate. Ubiquitin is usually attached to lysine residues and less frequently to the N terminus of proteins. In addition, several viral E3s have been identified that attach ubiquitin to cysteine or serine/threonine residues. Mutation of lysines, serines, and threonines in the NS-1 variable region was necessary to significantly reduce ubiquitylation and stabilize the protein. The Hrd1 E3 ligase was required to modify all three amino acids. Our studies argue that ubiquitylation of ER proteins relies on very different mechanisms of recognition and modification than those used to regulate bio! logical processes.
• 3D Cryo-EM Structure of an Active Step I Spliceosome and Localization of Its Catalytic Core
  - mol cell 40(6):927-938 (2010)
  The spliceosome excises introns from pre-mRNA in a two-step splicing reaction. So far, the three-dimensional (3D) structure of a spliceosome with preserved catalytic activity has remained elusive. Here, we determined the 3D structure of the human, catalytically active step I spliceosome (C complex) by cryo-electron microscopy (cryo-EM) in vitrified ice. Via immunolabeling we mapped the position of the 5′ exon. The C complex contains an unusually salt-stable ribonucleoprotein (RNP) core that harbors its catalytic center. We determined the 3D structure of this RNP core and also that of a post-step II particle, the 35S U5 snRNP, which contains most of the C complex core proteins. As C complex domains could be recognized in these structures, their position in the C complex could be determined, thereby allowing the region harboring the spliceosome's catalytic core to be localized.
• Genome-wide Identification of Polycomb-Associated RNAs by RIP-seq
  - mol cell 40(6):939-953 (2010)
  Polycomb proteins play essential roles in stem cell renewal and human disease. Recent studies of HOX genes and X inactivation have provided evidence for RNA cofactors in Polycomb repressive complex 2 (PRC2). Here we develop a RIP-seq method to capture the PRC2 transcriptome and identify a genome-wide pool of >9000 PRC2-interacting RNAs in embryonic stem cells. The transcriptome includes antisense, intergenic, and promoter-associated transcripts, as well as many unannotated RNAs. A large number of transcripts occur within imprinted regions, oncogene and tumor suppressor loci, and stem cell-related bivalent domains. We provide evidence for direct RNA-protein interactions, most likely via the Ezh2 subunit. We also identify Gtl2 RNA as a PRC2 cofactor that directs PRC2 to the reciprocally imprinted Dlk1 coding gene. Thus, Polycomb proteins interact with a genome-wide family of RNAs, some of which may be used as biomarkers and therapeutic targets for human disease.
• A Transcriptional Activator Is Part of an SCF Ubiquitin Ligase to Control Degradation of Its Cofactors
  - mol cell 40(6):954-964 (2010)
  Multisubunit protein complexes pose a challenge to the coordinated regulation of individual components. We show how the yeast transactivating factor Met4 functions as a component of the SCFMet30 ubiquitin ligase to synchronize its own activity with cofactor assembly. Cells maintain Met4 in a dormant state by a regulatory ubiquitin chain assembled by SCFMet30. Nutritional and heavy-metal stress block Met4 ubiquitylation resulting in Met4 activation, which induces a stress-response program including cell-cycle arrest. Met4 relies on assembly with various cofactors for promoter binding. We report here that the stability of these DNA-binding cofactors is regulated by SCFMet30. Remarkably, the transcriptional activator Met4 functions as a substrate-specificity factor in the context of SCFMet30/Met4 to coordinate cofactor degradation with its own activity status. Our results establish an additional layer for substrate recruitment by SCF ubiquitin ligases and provide conceptu! al insight into coordinated regulation of protein complexes.
• Recruitment Timing and Dynamics of Transcription Factors at the Hsp70 Loci in Living Cells
  - mol cell 40(6):965-975 (2010)
  Chromatin immunoprecipitation (ChIP) studies provide snapshots of factors on chromatin in cell populations. Here, we use live-cell imaging to examine at high temporal resolution the recruitment and dynamics of transcription factors to the inducible Hsp70 loci in individual Drosophila salivary gland nuclei. Recruitment of the master regulator, HSF, is first detected within 20 s of gene activation; the timing of its recruitment resolves from RNA polymerase II and P-TEFb, and these factors resolve from Spt6 and Topo I. Remarkably, the recruitment of each factor is highly synchronous between different cells. In addition, fluorescence recovery after photobleaching (FRAP) analyses show that the entry and exit of multiple factors are progressively constrained upon gene activation, suggesting the gradual formation of a transcription compartment. Furthermore, we demonstrate that poly(ADP-ribose) (PAR) polymerase activity is required to maintain the transcription compartment. We! propose that PAR polymers locally retain factors in a transcription compartment.
• The ACF1 Complex Is Required for DNA Double-Strand Break Repair in Human Cells
  - mol cell 40(6):976-987 (2010)
  DNA double-strand breaks (DSBs) are repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR), but cellular repair processes remain elusive. We show here that the ATP-dependent chromatin-remodeling factors, ACF1 and SNF2H, accumulate rapidly at DSBs and are required for DSB repair in human cells. If the expression of ACF1 or SNF2H is suppressed, cells become extremely sensitive to X-rays and chemical treatments producing DSBs, and DSBs remain unrepaired. ACF1 interacts directly with KU70 and is required for the accumulation of KU proteins at DSBs. The KU70/80 complex becomes physically more associated with the chromatin-remodeling factors of the CHRAC complex, which includes ACF1, SNF2H, CHRAC15, and CHRAC17, after treatments producing DSBs. Furthermore, the frequency of NHEJ as well as HR induced by DSBs in chromosomal DNA is significantly decreased in cells depleted of either of these factors. Thus, ACF1 and its complexes play important roles in ! DSBs repair.
• Mus81 and Yen1 Promote Reciprocal Exchange during Mitotic Recombination to Maintain Genome Integrity in Budding Yeast
  - mol cell 40(6):988-1000 (2010)
  Holliday junction (HJ) resolution is required for segregation of chromosomes and for formation of crossovers during homologous recombination. The identity of the resolvase(s) that functions in vivo has yet to be established, although several proteins able to cut HJs in vitro have been identified as candidates in yeasts and mammals. Using an assay to detect unselected products of mitotic recombination, we found a significant decrease in crossovers in the Saccharomyces cerevisiae mus81Δ mutant. Yen1 serves a backup function responsible for resolving intermediates in mus81Δ mutants, or when conversion tracts are short. In the absence of both Mus81 and Yen1, intermediates are not channeled exclusively to noncrossover recombinants, but instead are processed by Pol32-dependent break-induced replication (BIR). The channeling of recombination from reciprocal exchange to BIR results in greatly increased spontaneous loss of heterozygosity (LOH) and chromosome mis-segregation i! n the mus81Δ yen1Δ mutant, typical of the genomic instability found in tumor cells.
• Temporally and Biochemically Distinct Activities of Exo1 during Meiosis: Double-Strand Break Resection and Resolution of Double Holliday Junctions
  - mol cell 40(6):1001-1015 (2010)
  The Rad2/XPG family nuclease, Exo1, functions in a variety of DNA repair pathways. During meiosis, Exo1 promotes crossover recombination and thereby facilitates chromosome segregation at the first division. Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs). Nucleolytic resection of DSBs generates long 3′ single-strand tails that undergo strand exchange with a homologous chromosome to form joint molecule (JM) intermediates. We show that meiotic DSB resection is dramatically reduced in exo1Δ mutants and test the idea that Exo1-catalyzed resection promotes crossing over by facilitating formation of crossover-specific JMs called double Holliday junctions (dHJs). Contrary to this idea, dHJs form at wild-type levels in exo1Δ mutants, implying that Exo1 has a second function that promotes resolution of dHJs into crossovers. Surprisingly, the dHJ resolution function of Exo1 is independent of its nuclease activities but requires interaction wi! th the putative endonuclease complex, Mlh1-Mlh3. Thus, the DSB resection and procrossover functions of Exo1 during meiosis involve temporally and biochemically distinct activities.
• TDRD3 Is an Effector Molecule for Arginine-Methylated Histone Marks
  - mol cell 40(6):1016-1023 (2010)
  Specific sites of histone tail methylation are associated with transcriptional activity at gene loci. These methyl marks are interpreted by effector molecules, which harbor protein domains that bind the methylated motifs and facilitate either active or inactive states of transcription. CARM1 and PRMT1 are transcriptional coactivators that deposit H3R17me2a and H4R3me2a marks, respectively. We used a protein domain microarray approach to identify the Tudor domain-containing protein TDRD3 as a "reader" of these marks. Importantly, TDRD3 itself is a transcriptional coactivator. This coactivator activity requires an intact Tudor domain. TDRD3 is recruited to an estrogen-responsive element in a CARM1-dependent manner. Furthermore, ChIP-seq analysis of TDRD3 reveals that it is predominantly localized to transcriptional start sites. Thus, TDRD3 is an effector molecule that promotes transcription by binding methylarginine marks on histone tails.