Hot off the presses! Dec 24 Mol Cell

The Dec 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:

• Weizmann Young PI Forum: The Power of Peer Support
  - Mol Cell 36(6):913-915 (2009)
  The academic path is a challenging journey full of hurdles and without a clear roadmap. As young faculty, we searched for support in steering through the complexities of our new roles. Here we describe our experience in forming a peer support group and share the lessons learnt along the way.
• FANCM: A Landing Pad for the Fanconi Anemia and Bloom's Syndrome Complexes
  - Mol Cell 36(6):916-917 (2009)
  In this issue of Molecular Cell, Deans and West (2009) reveal the molecular basis of the phenotypic similarities between Fanconi Anemia (FA) and Bloom's Syndrome, identifying FANCM as the anchor for both FA and Bloom's complexes at the site of the DNA interstrand crosslink.
• RNA Processing: Redrawing the Map of Charted Territory
  - Mol Cell 36(6):918-919 (2009)
  Using genome-wide RNA-binding data, Xue et al. (2009) draw a regulatory map in this issue of Molecular Cell for the much-studied polypyrimidine tract-binding protein (PTB) that reveals a unique paradigm in posttranscriptional gene regulation.
• HuD Stimulates Translation via eIF4A
  - Mol Cell 36(6):920-921 (2009)
  In this issue of Molecular Cell, Fukao et al. (2009) report that HuD upregulates mRNA translation through direct interaction with eIF4A in the 5′ cap-binding complex, revealing a posttranscriptional role for HuD in neuronal development and plasticity.
• Rhomboid Proteases: Familiar Features in Unfamiliar Phases
  - Mol Cell 36(6):922-923 (2009)
  In this issue of Molecular Cell, Strisovsky et al. (2009) identify a sequence motif underlying cleavage site specificity for the rhomboid proteases. This sheds light on potential mechanisms by which intramembrane-cleaving proteases cleave their substrates.
• Shifting Players and Paradigms in Cell-Specific Transcription
  - Mol Cell 36(6):924-931 (2009)
  Historically, developmental-stage- and tissue-specific patterns of gene expression were assumed to be determined primarily by DNA regulatory sequences and their associated activators, while the general transcription machinery including core promoter recognition complexes, coactivators, and chromatin modifiers was held to be invariant. New evidence suggests that significant changes in these general transcription factors including TFIID, BAF, and Mediator may facilitate global changes in cell-type-specific transcription.
• Eukaryotic Stress Granules: The Ins and Outs of Translation
  - Mol Cell 36(6):932-941 (2009)
  The stress response in eukaryotic cells often inhibits translation initiation and leads to the formation of cytoplasmic RNA-protein complexes referred to as stress granules. Stress granules contain nontranslating mRNAs, translation initiation components, and many additional proteins affecting mRNA function. Stress granules have been proposed to affect mRNA translation and stability and have been linked to apoptosis and nuclear processes. Stress granules also interact with P-bodies, another cytoplasmic RNP granule containing nontranslating mRNA, translation repressors, and some mRNA degradation machinery. Together, stress granules and P-bodies reveal a dynamic cycle of distinct biochemical and compartmentalized mRNPs in the cytosol, with implications for the control of mRNA function.
• FANCM Connects the Genome Instability Disorders Bloom's Syndrome and Fanconi Anemia
  - Mol Cell 36(6):943-953 (2009)
  Fanconi Anemia (FA) and Bloom's Syndrome (BS) are genetic disorders characterized by overlapping phenotypes, including aberrant DNA repair and cancer predisposition. Here, we show that the FANCM gene product, FANCM protein, links FA and BS by acting as a protein anchor and bridge that targets key components of the FA and BS pathways to stalled replication forks, thus linking multiple components that are necessary for efficient DNA repair. Two highly conserved protein:protein interaction motifs in FANCM, designated MM1 and MM2, were identified. MM1 interacts with the FA core complex by binding to FANCF, whereas MM2 interacts with RM1 and topoisomerase IIIα, components of the BS complex. The MM1 and MM2 motifs were independently required to activate the FA and BS pathways. Moreover, a common phenotype of BS and FA cells—an elevated frequency of sister chromatid exchanges—was due to a loss of interaction of the two complexes through FANCM.
• CtIP Links DNA Double-Strand Break Sensing to Resection
  - Mol Cell 36(6):954-969 (2009)
  In response to DNA double-strand breaks (DSBs), cells sense the DNA lesions and then activate the protein kinase ATM. Subsequent DSB resection produces RPA-coated ssDNA that is essential for activation of the DNA damage checkpoint and DNA repair by homologous recombination (HR). However, the biochemical mechanism underlying the transition from DSB sensing to resection remains unclear. Using Xenopus egg extracts and human cells, we show that the tumor suppressor protein CtIP plays a critical role in this transition. We find that CtIP translocates to DSBs, a process dependent on the DSB sensor complex Mre11-Rad50-NBS1, the kinase activity of ATM, and a direct DNA-binding motif in CtIP, and then promotes DSB resection. Thus, CtIP facilitates the transition from DSB sensing to processing: it does so by binding to the DNA at DSBs after DSB sensing and ATM activation and then promoting DNA resection, leading to checkpoint activation and HR.
• A Reconfigured Pattern of MLL Occupancy within Mitotic Chromatin Promotes Rapid Transcriptional Reactivation Following Mitotic Exit
  - Mol Cell 36(6):970-983 (2009)
  Mixed lineage leukemia (MLL) and its metazoan Trithorax orthologs have been linked with the epigenetic maintenance of transcriptional activity. To identify mechanisms by which MLL perpetuates active transcription in dividing cells, we investigated its role during M phase of the cell cycle. Unlike other chromatin-modifying enzymes examined, we found that MLL associates with gene promoters packaged within condensed mitotic chromosomes. Genome-wide location analysis identified a globally rearranged pattern of MLL occupancy during mitosis in a manner favoring genes that were highly transcribed during interphase. Knockdown experiments revealed that MLL retention at gene promoters during mitosis accelerates transcription reactivation following mitotic exit. MLL tethers Menin, RbBP5, and ASH2L to its occupied sites during mitosis, but is dispensable for preserving histone H3K4 methylation. These findings implicate mitotic bookmarking as a component of Trithorax-based gene reg! ulation, which may facilitate inheritance of active gene expression states during cell division.
• Controlling Hematopoiesis through Sumoylation-Dependent Regulation of a GATA Factor
  - Mol Cell 36(6):984-995 (2009)
  GATA factors establish transcriptional networks that control fundamental developmental processes. Whereas the regulator of hematopoiesis GATA-1 is subject to multiple posttranslational modifications, how these modifications influence GATA-1 function at endogenous loci is unknown. We demonstrate that sumoylation of GATA-1 K137 promotes transcriptional activation only at target genes requiring the coregulator Friend of GATA-1 (FOG-1). A mutation of GATA-1 V205G that disrupts FOG-1 binding and K137 mutations yielded similar phenotypes, although sumoylation was FOG-1 independent, and FOG-1 binding did not require sumoylation. Both mutations dysregulated GATA-1 chromatin occupancy at select sites, FOG-1-dependent gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, FOG-1- and SUMO-1-independent genes persisted at the periphery. These results illustrate a mechanism t! hat controls trans-acting factor function in a locus-specific manner, and differentially regulated members of the target gene ensemble reside in distinct subnuclear compartments.
• Genome-wide Analysis of PTB-RNA Interactions Reveals a Strategy Used by the General Splicing Repressor to Modulate Exon Inclusion or Skipping
  - Mol Cell 36(6):996-1006 (2009)
  Recent transcriptome analysis indicates that > 90% of human genes undergo alternative splicing, underscoring the contribution of differential RNA processing to diverse proteomes in higher eukaryotic cells. The polypyrimidine tract-binding protein PTB is a well-characterized splicing repressor, but PTB knockdown causes both exon inclusion and skipping. Genome-wide mapping of PTB-RNA interactions and construction of a functional RNA map now reveal that dominant PTB binding near a competing constitutive splice site generally induces exon inclusion, whereas prevalent binding close to an alternative site often causes exon skipping. This positional effect was further demonstrated by disrupting or creating a PTB-binding site on minigene constructs and testing their responses to PTB knockdown or overexpression. These findings suggest a mechanism for PTB to modulate splice site competition to produce opposite functional consequences, which may be generally applicable to RNA-bin! ding splicing factors to positively or negatively regulate alternative splicing in mammalian cells.
• The ELAV Protein HuD Stimulates Cap-Dependent Translation in a Poly(A)- and eIF4A-Dependent Manner
  - Mol Cell 36(6):1007-1017 (2009)
  The RNA-binding protein HuD promotes neuronal differentiation by an unknown mechanism. Here we identify an enhancer function of HuD in translation. Translation stimulation by HuD requires both a 3′ poly(A) tail and a 5′ m7G cap structure. We also show that HuD directly interacts with eIF4A. This interaction and the poly(A)-binding activity of HuD are critical for its translational enhancer function because HuD-eIF4A- and HuD-poly(A)-binding mutants fail to stimulate translation. We show that translation of HCV IRES mRNA, which is eIF4A independent, is not stimulated by HuD. We also find that the eIF4A and poly(A)-binding activities of HuD are not only important for stimulating translation but also are essential for HuD-induced neurite outgrowth in PC12 cells. This example of cap-dependent translational regulation might explain at least in part how HuD triggers the induction of neuronal differentiation.
• Together, Rpn10 and Dsk2 Can Serve as a Polyubiquitin Chain-Length Sensor
  - Mol Cell 36(6):1018-1033 (2009)
  As a signal for substrate targeting, polyubiquitin meets various layers of receptors upstream to the 26S proteasome. We obtained structural information on two receptors, Rpn10 and Dsk2, alone and in complex with (poly)ubiquitin or with each other. A hierarchy of affinities emerges with Dsk2 binding monoubiquitin tighter than Rpn10 does, whereas Rpn10 prefers the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. Dynamic rearrangement provides a chain-length sensor, possibly explaining how accessibility of Dsk2 to the proteasome is limited unless it carries a properly tagged cargo. We propose a mechanism for a malleable ubiquitin signal that depends both on ch! ain length and combination of receptors to produce tetraubiquitin as an efficient signal threshold.
• SH3 Domains from a Subset of BAR Proteins Define a Ubl-Binding Domain and Implicate Parkin in Synaptic Ubiquitination
  - Mol Cell 36(6):1034-1047 (2009)
  Mutations in the parkin gene are responsible for a common inherited form of Parkinson's disease (PD). Parkin is a RING-type E3 ubiquitin ligase with an N-terminal ubiquitin-like domain (Ubl). We report here that the parkin Ubl binds SH3 domains from endocytic BAR proteins such as endophilin-A with an affinity comparable to proline-rich domains (PRDs) from well-established SH3 partners. The NMR structure of the Ubl-SH3 complex identifies the PaRK extension, a unique C-terminal motif in the parkin Ubl required for SH3 binding and for parkin-mediated ubiquitination of endophilin-A in vitro. In nerve terminals, conditions that promote phosphorylation enhance the interaction between parkin and endophilin-A and increase the levels of ubiquitinated proteins within PRD-associated synaptic protein complexes in wild-type but not parkin knockout brain. The findings identify a pathway for the recruitment of synaptic substrates to parkin with the potential to explain the defects in! synaptic transmission observed in recessive forms of PD.
• Sequence-Specific Intramembrane Proteolysis: Identification of a Recognition Motif in Rhomboid Substrates
  - Mol Cell 36(6):1048-1059 (2009)
  Members of the widespread rhomboid family of intramembrane proteases cleave transmembrane domain (TMD) proteins to regulate processes as diverse as EGF receptor signaling, mitochondrial dynamics, and invasion by apicomplexan parasites. However, lack of information about their substrates means that the biological role of most rhomboids remains obscure. Knowledge of how rhomboids recognize their substrates would illuminate their mechanism and might also allow substrate prediction. Previous work has suggested that rhomboid substrates are specified by helical instability in their TMD. Here we demonstrate that rhomboids instead primarily recognize a specific sequence surrounding the cleavage site. This recognition motif is necessary for substrate cleavage, it determines the cleavage site, and it is more strictly required than TM helix-destabilizing residues. Our work demonstrates that intramembrane proteases can be sequence specific and that genome-wide substrate prediction! based on their recognition motifs is feasible.
• RabGDI Displacement by DrrA from Legionella Is a Consequence of Its Guanine Nucleotide Exchange Activity
  - Mol Cell 36(6):1060-1072 (2009)
  Prenylated Rab proteins exist in the cytosol as soluble, high-affinity complexes with GDI that need to be disrupted for membrane attachment and targeting of Rab proteins. The Legionella pneumophila protein DrrA displaces GDI from Rab1:GDI complexes, incorporating Rab1 into Legionella-containing vacuoles and activating Rab1 by exchanging GDP for GTP. Here, we present the crystal structure of a complex between the GEF domain of DrrA and Rab1 and a detailed kinetic analysis of this exchange. DrrA efficiently catalyzes nucleotide exchange and mimics the general nucleotide exchange mechanism of mammalian GEFs for Ras-like GTPases. We show that the GEF activity of DrrA is sufficient to displace prenylated Rab1 from the Rab1:GDI complex. Thus, apparent GDI displacement by DrrA is linked directly to nucleotide exchange, suggesting a basic model for GDI displacement and specificity of Rab localization that does not require discrete GDI displacement activity.
• A Model of Partnership Co-Opted by the Homeodomain Protein TGIF and the Itch/AIP4 Ubiquitin Ligase for Effective Execution of TNF-α Cytotoxicity
  - Mol Cell 36(6):1073-1085 (2009)
  The homeodomain protein TGIF functions as a negative regulator of multiple classes of transcription factors. Here we report on the characterization of TGIF as an essential component of the tumor necrosis factor alpha (TNF-α) cytotoxic program. This proapoptotic role of TGIF does not appear to rely on transcriptional modulation but instead is executed in conjunction with Itch/AIP4, an E3 ubiquitin ligase operating in TNF-α-induced apoptosis through its ability to target the caspase antagonist cFlipL for degradation. Notably, we found that activation of TNF-α signaling induced the association of TGIF with Itch/AIP4, resulting in increased accessibility of cFlipL for association and ubiquitination by Itch/AIP4. Moreover, we show that Itch/AIP4 can also stabilize the TGIF protein in response to TNF-α by triggering its monoubiquitination at lysine 259, thereby revealing the existence of a functional network that can evolve into a positive feedback loop for ensuring effe! ctive execution of the TNF-α apoptotic signaling.
• Nucleosome Remodeling by hMSH2-hMSH6
  - Mol Cell 36(6):1086-1094 (2009)
  DNA nucleotide mismatches and lesions arise on chromosomes that are a complex assortment of protein and DNA (chromatin). The fundamental unit of chromatin is a nucleosome that contains 146 bp DNA wrapped around an H2A, H2B, H3, and H4 histone octamer. We demonstrate that the mismatch recognition heterodimer hMSH2-hMSH6 disassembles a nucleosome. Disassembly requires a mismatch that provokes the formation of hMSH2-hMSH6 hydrolysis-independent sliding clamps, which translocate along the DNA to the nucleosome. The rate of disassembly is enhanced by actual or mimicked acetylation of histone H3 within the nucleosome entry-exit and dyad axis that occurs during replication and repair in vivo and reduces DNA-octamer affinity in vitro. Our results support a passive mechanism for chromatin remodeling whereby hMSH2-hMSH6 sliding clamps trap localized fluctuations in nucleosome positioning and/or wrapping that ultimately leads to disassembly, and highlight unanticipated strengths ! of the Molecular Switch Model for mismatch repair (MMR).
• Insights into Ubiquitin Transfer Cascades from a Structure of a UbcH5BUbiquitin-HECTNEDD4L Complex
  - Mol Cell 36(6):1095-1102 (2009)
  In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2Ub covalent complex and then interacts with an E3 for Ub transfer. For cascades involving E3s in the HECT class, Ub is transferred from an associated E2 to the acceptor cysteine in the HECT domain C lobe. To gain insights into this process, we determined the crystal structure of a complex between the HECT domain of NEDD4L and the E2 UbcH5B bearing a covalently linked Ub at its active site (UbcH5BUb). Noncovalent interactions between UbcH5B and the HECT N lobe and between Ub and the HECT domain C lobe lead to an overall compact structure, with the Ub C terminus sandwiched between UbcH5B and HECT domain active sites. The structure suggests a model for E2-to-HECT Ub transfer, in which interactions between a donor Ub and an acceptor domain constrain upstream and downstream enzymes for conjugation.