Hot off the presses! Nov 24 mol cell

The Nov 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:

• ATM: Promoter of Metabolic "Cost" Reduction and "Savings" Usage during Hypoxia through mTORC1 Regulation
  - mol cell 40(4):501-502 (2010)
  Cells must sense environmental conditions and adjust to maintain metabolic homeostasis and survive stress conditions; in this issue, Cam et al. (2010) show that the tumor suppressor kinase ATM is activated by hypoxia, phosphorylates and stabilizes HIF-1α, and inhibits mTORC1.
• A Pause to Splice
  - mol cell 40(4):503-505 (2010)
  Maturation of mRNA termini occurs during transcription and can be aided by pausing of RNA polymerase II (RNAPII). In this issue of Molecular Cell, two groups now demonstrate that RNAPII pausing may also assist cotranscriptional splicing in S. cerevisiae.
• Choreography of the 9-1-1 Checkpoint Complex: DDK Puts a Check on the Checkpoints
  - mol cell 40(4):505-506 (2010)
  Checkpoint proteins respond to DNA damage by halting the cell cycle until the damage is repaired. In this issue of Molecular Cell, Furuya et al. (2010) provide evidence that checkpoint proteins need to be removed from sites of damage in order to properly repair it.
• The Meaning of Pausing
  - mol cell 40(4):507-508 (2010)
  Pausing of RNA polymerase II (RNAPII) at the 5′ end of genes is a widespread phenomenon in metazoans, but the role of this event in gene regulation is poorly understood. Gilchrist et al. (2010) now demonstrate that RNAPII pausing counteracts DNA-influenced nucleosome organization to allow precise gene activation.
• mTORC1 Signaling under Hypoxic Conditions Is Controlled by ATM-Dependent Phosphorylation of HIF-1α
  - mol cell 40(4):509-520 (2010)
  The mTOR complex-1 (mTORC1) coordinates cell growth and metabolism, acting as a restriction point under stress conditions such as low oxygen tension (hypoxia). Hypoxia suppresses mTORC1 signaling. However, the signals by which hypoxia suppresses mTORC1 are only partially understood, and a direct link between hypoxia-driven physiological stress and the regulation of mTORC1 signaling is unknown. Here we show that hypoxia results in ataxia telangiectasia mutated (ATM)-dependent phosphorylation of hypoxia-inducible factor 1-alpha (HIF-1α) on serine696 and mediates downregulation of mTORC1 signaling. Deregulation of these pathways in pediatric solid tumor xenografts suggests a link between mTORC1 dysregulation and solid tumor development and points to an important role for hypoxic regulation of mTORC1 activity in tumor development.
• PARP-1 Attenuates Smad-Mediated Transcription
  - mol cell 40(4):521-532 (2010)
  The versatile cytokine transforming growth factor β (TGF-β) regulates cellular growth, differentiation, and migration during embryonic development and adult tissue homeostasis. Activation of TGF-β receptors leads to phosphorylation of Smad2 and Smad3, which oligomerize with Smad4 and accumulate in the nucleus where they recognize gene regulatory regions and orchestrate transcription. Termination of Smad-activated transcription involves Smad dephosphorylation, nuclear export, or ubiquitin-mediated degradation. In an unbiased proteomic screen, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a Smad-interacting partner. PARP-1 dissociates Smad complexes from DNA by ADP-ribosylating Smad3 and Smad4, which attenuates Smad-specific gene responses and TGF-β-induced epithelial-mesenchymal transition. Thus, our results identify ADP-ribosylation of Smad proteins by PARP-1 as a key step in controlling the strength and duration of Smad-mediated transcription.
• Primary Cilium-Dependent and -Independent Hedgehog Signaling Inhibits p16INK4A
  - mol cell 40(4):533-547 (2010)
  In a genome-wide siRNA analysis of p16INK4a (p16) modulators, we identify the Hedgehog (Hh) pathway component SUFU and formally demonstrate that Hh signaling promotes mitogenesis by suppression of p16. A fragment of the Hh-responsive GLI2 transcription factor directly binds and inhibits the p16 promoter and senescence is associated with the loss of nuclear GLI2. Hh components partially reside in the primary cilium (PC), and the small fraction of cells in mass culture that elaborate a PC have the lowest expression of p16. Suppression of p16 is effected by both PC-dependent and -independent routes, and ablation of p16 renders cells insensitive to an Hh inhibitor and increases PC formation. These results directly link a well-established developmental mitogenic pathway with a key tumor suppressor and contribute to the molecular understanding of replicative senescence, Hh-mediated oncogenesis, and potentially the role of p16 in aging.
• Ubiquitin Binding to A20 ZnF4 Is Required for Modulation of NF-κB Signaling
  - mol cell 40(4):548-557 (2010)
  Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger 4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to t! he Ub editing process and, ultimately, to regulation of NF-κB signaling.
• Pervasive and Cooperative Deadenylation of 3′UTRs by Embryonic MicroRNA Families
  - mol cell 40(4):558-570 (2010)
  To understand how miRNA-mediated silencing impacts on embryonic mRNAs, we conducted a functional survey of abundant maternal and zygotic miRNA families in the C. elegans embryo. We show that the miR-35-42 and the miR-51-56 miRNA families define maternal and zygotic miRNA-induced silencing complexes (miRISCs), respectively, that share a large number of components. Using a cell-free C. elegans embryonic extract, we demonstrate that the miRISC directs the rapid deadenylation of reporter mRNAs with natural 3′UTRs. The deadenylated targets are translationally suppressed and remarkably stable. Sampling of the predicted miR-35-42 targets reveals that roughly half are deadenylated in a miRNA-dependent manner, but with each target displaying a distinct efficiency and pattern of deadenylation. Finally, we demonstrate that functional cooperation between distinct miRISCs within 3′UTRs is required to potentiate deadenylation. With this report, we reveal the extensive and direct! impact of miRNA-mediated deadenylation on embryonic mRNAs.
• Global Analysis of Nascent RNA Reveals Transcriptional Pausing in Terminal Exons
  - mol cell 40(4):571-581 (2010)
  Pre-mRNA splicing is catalyzed by the spliceosome, which can assemble on pre-mRNA cotranscriptionally. However, whether splicing generally occurs during transcription has not been addressed. Indeed, splicing catalysis is expected to occur posttranscriptionally in yeast, where the shortness of terminal exons should leave insufficient time for splicing. Here, we isolate endogenous S. cerevisiae nascent RNA and determine gene-specific splicing efficiencies and transcription profiles, using high-density tiling microarrays. Surprisingly, we find that splicing occurs cotranscriptionally for the majority of intron-containing genes. Analysis of transcription profiles reveals Pol II pausing within the terminal exons of these genes. Intronless and inefficiently spliced genes lack this pause. In silico simulations of transcription and splicing kinetics confirm that this pausing event provides sufficient time for splicing before termination. The discovery of terminal exon pausing ! demonstrates functional coupling of transcription and splicing near gene ends.
• Splicing-Dependent RNA Polymerase Pausing in Yeast
  - mol cell 40(4):582-593 (2010)
  In eukaryotic cells, there is evidence for functional coupling between transcription and processing of pre-mRNAs. To better understand this coupling, we performed a high-resolution kinetic analysis of transcription and splicing in budding yeast. This revealed that shortly after induction of transcription, RNA polymerase accumulates transiently around the 3′ end of the intron on two reporter genes. This apparent transcriptional pause coincides with splicing factor recruitment and with the first detection of spliced mRNA and is repeated periodically thereafter. Pausing requires productive splicing, as it is lost upon mutation of the intron and restored by suppressing the splicing defect. The carboxy-terminal domain of the paused polymerase large subunit is hyperphosphorylated on serine 5, and phosphorylation of serine 2 is first detected here. Phosphorylated polymerase also accumulates around the 3′ splice sites of constitutively expressed, endogenous yeast genes. We! propose that transcriptional pausing is imposed by a checkpoint associated with cotranscriptional splicing.
• Jmjd3 and UTX Play a Demethylase-Independent Role in Chromatin Remodeling to Regulate T-Box Family Member-Dependent Gene Expression
  - mol cell 40(4):594-605 (2010)
  The stable and heritable H3K27-methyl mark suppresses transcription of lineage-specific genes in progenitor cells. During developmental transitions, histone demethylases are required to dramatically alter epigenetic and gene expression states to create new cell-specific profiles. It is unclear why demethylase proteins that antagonize polycomb-mediated repression continue to be expressed in terminally differentiated cells where further changes in H3K27 methylation could be deleterious. In this study, we show that the H3K27 demethylases, Jmjd3 and UTX, mediate a functional interaction between the lineage-defining T-box transcription factor family and a Brg1-containing SWI/SNF remodeling complex. Importantly, Jmjd3 is required for the coprecipitation of Brg1 with the T-box factor, T-bet, and this interaction is necessary for Ifng remodeling in differentiated Th1 cells. Thus, Jmjd3 has a required role in general chromatin remodeling that is independent from its H3K27 demet! hylase potential. This function for H3K27 demethylase proteins may explain their presence in differentiated cells where the epigenetic profile is already established.
• DDK Phosphorylates Checkpoint Clamp Component Rad9 and Promotes Its Release from Damaged Chromatin
  - mol cell 40(4):606-618 (2010)
  When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1Cdc7, the Schizosaccharomyces pombe Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5TopBP1, and prior phosphorylation by Rad3ATR. rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to! facilitate DNA repair.
• The MMS22L-TONSL Complex Mediates Recovery from Replication Stress and Homologous Recombination
  - mol cell 40(4):619-631 (2010)
  Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate ! the recombination-dependent repair of stalled or collapsed replication forks.
• Identification of the MMS22L-TONSL Complex that Promotes Homologous Recombination
  - mol cell 40(4):632-644 (2010)
  Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NFκBIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S phase-associated DSBs, such as topoisomerase (TOP) inhibitors. In this light, MMS22L and TONSL are required for the HR-mediated repair of replication fork-associated DSBs. In cells depleted of either protein, DSBs induced by the TOP1 inhibitor camptothecin are resected normally, but the loading of the RAD51 recombinase is defective. Therefore, MMS22L and TONSL are required for the maintenance of genome stability when unscheduled DSBs occur in the vicinity of DNA replication forks.
• A Genome-wide Camptothecin Sensitivity Screen Identifies a Mammalian MMS22L-NFKBIL2 Complex Required for Genomic Stability
  - mol cell 40(4):645-657 (2010)
  Replication stress involving collision of replisomes with camptothecin (CPT)-stabilized DNA-Topoisomerase I adducts activates an ATR-dependent pathway to promote repair by homologous recombination. To identify human genes that protect cells from such replication stress, we performed a genome-wide CPT sensitivity screen. Among numerous candidate genes are two previously unstudied proteins: the ankyrin repeat protein NFKBIL2 and C6ORF167 (MMS22L), distantly related to yeast replication stress regulator Mms22p. MMS22L and NFKBIL2 interact with each other and with FACT (facilitator of chromatin transcription) and MCM (minichromosome maintenance) complexes. Cells depleted of NFKBIL2 or MMS22L are sensitive to DNA-damaging agents, load phosphorylated RPA onto chromatin in a CTIP-dependent manner, activate the ATR/ATRIP-CHK1 and double-strand break repair signaling pathways, and are defective in HR. This study identifies MMS22L-NFKBIL2 as components of the replication stress ! control pathway and provides a resource for discovery of additional components of this pathway.
• Crystal Structures of RNase H2 in Complex with Nucleic Acid Reveal the Mechanism of RNA-DNA Junction Recognition and Cleavage
  - mol cell 40(4):658-670 (2010)
  Two classes of RNase H hydrolyze RNA of RNA/DNA hybrids. In contrast to RNase H1 that requires four ribonucleotides for cleavage, RNase H2 can nick duplex DNAs containing a single ribonucleotide, suggesting different in vivo substrates. We report here the crystal structures of a type 2 RNase H in complex with substrates containing a (5′)RNA-DNA(3′) junction. They revealed a unique mechanism of recognition and substrate-assisted cleavage. A conserved tyrosine residue distorts the nucleic acid at the junction, allowing the substrate to function in catalysis by participating in coordination of the active site metal ion. The biochemical and structural properties of RNase H2 explain the preference of the enzyme for junction substrates and establish the structural and mechanistic differences with RNase H1. Junction recognition is important for the removal of RNA embedded in DNA and may play an important role in DNA replication and repair.
• ATP-Dependent Steps in the Binding of Ubiquitin Conjugates to the 26S Proteasome that Commit to Degradation
  - mol cell 40(4):671-681 (2010)
  Eukaryotic cells target proteins for degradation by the 26S proteasome by attaching a ubiquitin chain. Using a rapid assay, we analyzed the initial binding of ubiquitinated proteins to purified 26S particles as an isolated process at 4°C. Subunits Rpn10 and Rpn13 contribute equally to the high-affinity binding of ubiquitin chains, but in their absence, ubiquitin conjugates bind to another site with 4-fold lower affinity. Conjugate binding is stimulated 2- to 4-fold by binding of ATP or the nonhydrolyzable analog, ATPγS (but not ADP), to the 19S ATPases. Following this initial, reversible association, ubiquitin conjugates at 37°C become more tightly bound through a step that requires ATP hydrolysis and a loosely folded domain on the protein, but appears independent of ubiquitin. Unfolded or loosely folded polypeptides can inhibit this tighter binding. This commitment step precedes substrate deubiquitination and allows for selection of ubiquitinated proteins capable o! f being unfolded and efficiently degraded.