Hot off the presses! Jul 22 Mol Cell

The Jul 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:

• Sam68 Guest STARs in TNF-α Signaling
  - Mol Cell 43(2):157-158 (2011)
  In this issue of Molecular Cell, Ramakrishnan and Baltimore (2011) identify Sam68 as a TNFR1 docking protein required for proper activation of TNF-α signaling.
• A Network of Its Own: The Unique Interactome of the Hsp90 Cochaperone, Sba1/p23
  - Mol Cell 43(2):159-160 (2011)
  In this issue of Molecular Cell, Echtenkamp et al. (2011) show that the molecular chaperone Sba1/p23, thought to function primarily as a key modulator of the Hsp90 chaperone complex, also operates in its own sphere of influence outside of its obligations to Hsp90.
• Competing for the Clamp: Promoting RNA Polymerase Processivity and Managing the Transition from Initiation to Elongation
  - Mol Cell 43(2):161-163 (2011)
  Transcription elongation factor NusG/Spt5 spans the central cleft of RNA polymerase and functionally competes with transcription initiation factors. This work highlights the RNA polymerase clamp as a target for regulation and points to dynamic interactions between initiation and elongation machineries.
• Gimme Phospho-Serine Five! Capping Enzyme Guanylyltransferase Recognition of the RNA Polymerase II CTD
  - Mol Cell 43(2):163-165 (2011)
  In this issue of Molecular Cell, two papers on the structure of murine capping enzyme guanylyltransferase (Ghosh et al., 2011) and yeast studies of the recognition of the RNA polymerase II CTD (Schwer and Shuman, 2011) describe the mechanism of recruitment of the capping apparatus to nascent pre-mRNAs.
• Sam68 Is Required for Both NF-κB Activation and Apoptosis Signaling by the TNF Receptor
  - Mol Cell 43(2):167-179 (2011)
  The RNA-binding protein Sam68 is implicated in various cellular processes including RNA metabolism, apoptosis, and signal transduction. Here we identify a role of Sam68 in TNF-induced NF-κB activation and apoptosis. We found that Sam68 is recruited to the TNF receptor, and its deficiency dramatically reduces RIP recruitment and ubiquitylation. It also impairs cIAP1 recruitment and maintenance of recruited TRAF2 at the TNF receptor. In its absence, activation of the TAK1-IKK kinase complex is defective, greatly reducing signal transduction. Sam68 is also found as a part of the TNF-induced cytoplasmic caspase-8-FADD complex. RIP is not recruited to this complex in Sam68 knockout cells, and caspase activation is virtually absent. These findings delineate previously unknown functions for Sam68 in the TNF signaling pathway, where it acts as a signaling adaptor both in the membrane-associated complex I and in the cytoplasmic complex II, regulating both NF-κB activation and! apoptosis.
• NF-κB Induction of the SUMO Protease SENP2: A Negative Feedback Loop to Attenuate Cell Survival Response to Genotoxic Stress
  - Mol Cell 43(2):180-191 (2011)
  Activation of NF-κB, pivotal for immunity and oncogenesis, is tightly controlled by multiple feedback mechanisms. In response to DNA damage, SUMOylation of NEMO (NF-κB essential modulator) is critical for NF-κB activation; however, the SUMO proteases and feedback mechanisms involved remain unknown. Here we show that among the six known Sentrin/SUMO-specific proteases (SENPs), only SENP2 can efficiently associate with NEMO, deSUMOylate NEMO, and inhibit NF-κB activation induced by DNA damage. We further show that NF-κB induces SENP2 (and SENP1) transcription selectively in response to genotoxic stimuli, which involves ataxia telangiectasia mutated (ATM)-dependent histone methylation of SENP2 promoter κB regions and NF-κB recruitment. SENP2 null cells display biphasic NEMO SUMOylation and activation of IKK and NF-κB, and higher resistance to DNA damage-induced cell death. Our study establishes a self-attenuating feedback mechanism selective to DNA damage-induced ! signaling to limit NF-κB-dependent cell survival responses.
• ATR Autophosphorylation as a Molecular Switch for Checkpoint Activation
  - Mol Cell 43(2):192-202 (2011)
  The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust ch! eckpoint response.
• Reversible SUMOylation of TBL1-TBLR1 Regulates β-Catenin-Mediated Wnt Signaling
  - Mol Cell 43(2):203-216 (2011)
  Dysregulation of Wnt signaling has been implicated in tumorigenesis. The role of Transducin β-like proteins TBL1-TBLR1 in the promotion of Wnt/β-catenin-mediated oncogenesis has recently been emphasized; however, the molecular basis of activation of Wnt signaling by the corepressor TBL1-TBLR1 is incompletely understood. Here, we show that both TBL1 and TBLR1 are SUMOylated in a Wnt signaling-dependent manner, and that this modification is selectively reversed by SUMO-specific protease I (SENP1). SUMOylation dismissed TBL1-TBLR1 from the nuclear hormone receptor corepressor (NCoR) complex, increased recruitment of the TBL1-TBLR1-β-catenin complex to the promoter of Wnt target genes, and consequently led to activation of Wnt signaling. Conversely, SENP1 decreased formation of the TBL1-TBLR1-β-catenin complex, leading to inhibition of β-catenin-mediated transcription. Importantly, inhibition of SUMOylation significantly decreased the tumorigenicity of SW480 colon can! cer cells. Thus, our data reveal a mechanism for activation of Wnt signaling via the SUMOylation-dependent disassembly of the corepressor complex.
• The ClpS Adaptor Mediates Staged Delivery of N-End Rule Substrates to the AAA+ ClpAP Protease
  - Mol Cell 43(2):217-228 (2011)
  The ClpS adaptor delivers N-end rule substrates to ClpAP, an energy-dependent AAA+ protease, for degradation. How ClpS binds specific N-end residues is known in atomic detail and clarified here, but the delivery mechanism is poorly understood. We show that substrate binding is enhanced when ClpS binds hexameric ClpA. Reciprocally, N-end rule substrates increase ClpS affinity for ClpA6. Enhanced binding requires the N-end residue and a peptide bond of the substrate, as well as multiple aspects of ClpS, including a side chain that contacts the substrate α-amino group and the flexible N-terminal extension (NTE). Finally, enhancement also needs the N domain and AAA+ rings of ClpA, connected by a long linker. The NTE can be engaged by the ClpA translocation pore, but ClpS resists unfolding/degradation. We propose a staged-delivery model that illustrates how intimate contacts between the substrate, adaptor, and protease reprogram specificity and coordinate handoff from the ! adaptor to the protease.
• Global Functional Map of the p23 Molecular Chaperone Reveals an Extensive Cellular Network
  - Mol Cell 43(2):229-241 (2011)
  In parallel with evolutionary developments, the Hsp90 molecular chaperone system shifted from a simple prokaryotic factor into an expansive network that includes a variety of cochaperones. We have taken high-throughput genomic and proteomic approaches to better understand the abundant yeast p23 cochaperone Sba1. Our work revealed an unexpected p23 network that displayed considerable independence from known Hsp90 clients. Additionally, our data uncovered a broad nuclear role for p23, contrasting with the historical dogma of restricted cytosolic activities for molecular chaperones. Validation studies demonstrated that yeast p23 was required for proper Golgi function and ribosome biogenesis, and was necessary for efficient DNA repair from a wide range of mutagens. Notably, mammalian p23 had conserved roles in these pathways as well as being necessary for proper cell mobility. Taken together, our work demonstrates that the p23 chaperone serves a broad physiological network! and functions both in conjunction with and sovereign to Hsp90.
• Prion Induction by the Short-Lived, Stress-Induced Protein Lsb2 Is Regulated by Ubiquitination and Association with the Actin Cytoskeleton
  - Mol Cell 43(2):242-252 (2011)
  Yeast prions are self-perpetuating, QN-rich amyloids that control heritable traits and serve as a model for mammalian amyloidoses. De novo prion formation by overproduced prion protein is facilitated by other aggregated QN-rich protein(s) and is influenced by alterations of protein homeostasis. Here we explore the mechanism by which the Las17-binding protein Lsb2 (Pin3) promotes conversion of the translation termination factor Sup35 into its prion form, [PSI+]. We show that Lsb2 localizes with some Sup35 aggregates and that Lsb2 is a short-lived protein whose levels are controlled via the ubiquitin-proteasome system and are dramatically increased by stress. Loss of Lsb2 decreases stability of [PSI+] after brief heat shock. Mutations interfering with Lsb2 ubiquitination increase prion induction, while a mutation eliminating association of Lsb2 with the actin cytoskeleton blocks its aggregation and prion-inducing ability. These findings directly implicate the UPS and act! in cytoskeleton in regulating prions via a stress-inducible QN-rich protein.
• The β Subunit Gate Loop Is Required for RNA Polymerase Modification by RfaH and NusG
  - Mol Cell 43(2):253-262 (2011)
  In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the β gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the β′ clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity.
• The Initiation Factor TFE and the Elongation Factor Spt4/5 Compete for the RNAP Clamp during Transcription Initiation and Elongation
  - Mol Cell 43(2):263-274 (2011)
  TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP "clamp" region as a regulatory hot spot for both transcription initi! ation and transcription elongation.
• PHD Finger Recognition of Unmodified Histone H3R2 Links UHRF1 to Regulation of Euchromatic Gene Expression
  - Mol Cell 43(2):275-284 (2011)
  Histone methylation occurs on both lysine and arginine residues, and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD finger (PHDUHRF1), an important regulator of DNA CpG methylation, as a histone H3 unmodified arginine 2 (H3R2) recognition modality. This conclusion is based on binding studies and cocrystal structures of PHDUHRF1 bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1's ability to repress its direct target gene expression is dependent on PHDUHRF1 binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 functi! on.
• FGF-2 Regulates Cell Proliferation, Migration, and Angiogenesis through an NDY1/KDM2B-miR-101-EZH2 Pathway
  - Mol Cell 43(2):285-298 (2011)
  The histone H3K27 methyltransferase EZH2 plays an important role in oncogenesis, by mechanisms that are incompletely understood. Here, we show that the JmjC domain histone H3 demethylase NDY1 synergizes with EZH2 to silence the EZH2 inhibitor miR-101. NDY1 and EZH2 repress miR-101 by binding its promoter in concert, via a process triggered by upregulation of NDY1. Whereas EZH2 binding depends on NDY1, the latter binds independently of EZH2. However, both are required to repress transcription. NDY1 and EZH2 acting in concert upregulate EZH2 and stabilize the repression of miR-101 and its outcome. NDY1 is induced by FGF-2 via CREB phosphorylation and activation, downstream of DYRK1A, and mediates the FGF-2 and EZH2 effects on cell proliferation, migration, and angiogenesis. The FGF-2-NDY1/EZH2-miR-101-EZH2 axis described here was found to be active in bladder cancer. These data delineate an oncogenic pathway that functionally links FGF-2 with EZH2 via NDY1 and miR-101.
• Structural Insights to How Mammalian Capping Enzyme Reads the CTD Code
  - Mol Cell 43(2):299-310 (2011)
  Physical interaction between the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD) and cellular capping enzymes is required for efficient formation of the 5′ mRNA cap, the first modification of nascent mRNA. Here, we report the crystal structure of the RNA guanylyltransferase component of mammalian capping enzyme (Mce) bound to a CTD phosphopeptide. The CTD adopts an extended β-like conformation that docks Tyr1 and Ser5-PO4 onto the Mce nucleotidyltransferase domain. Structure-guided mutational analysis verified that the Mce-CTD interface is a tunable determinant of CTD binding and stimulation of guanylyltransferase activity, and of Mce function in vivo. The location and composition of the CTD binding site on mammalian capping enzyme is distinct from that of a yeast capping enzyme that recognizes the same CTD primary structure. Thus, capping enzymes from different taxa have evolved different strategies to read the CTD code.
• Deciphering the RNA Polymerase II CTD Code in Fission Yeast
  - Mol Cell 43(2):311-318 (2011)
  The RNA polymerase II carboxy-terminal domain (CTD) consists of tandem Y1S2P3T4S5P6S7 repeats. Dynamic remodeling of the CTD, especially its serine phosphorylation pattern, conveys informational cues about the transcription apparatus to a large ensemble of CTD-binding proteins. Our genetic dissection of fission yeast CTD function provides insights to the "CTD code." Two concepts stand out. First, the Ser2 requirement for transcription during sexual differentiation is bypassed by subtracting Ser7, signifying that imbalance in the phosphorylation array, not absence of a phospho-CTD cue, underlies a CTD-associated pathology. Second, the essentiality of Ser5 for vegetative growth is circumvented by covalently tethering mRNA capping enzymes to the CTD, thus proving that capping enzyme recruitment is a chief function of the Ser5-PO4 mark. This illustrates that a key "letter" in the CTD code can be neutralized by delivering its essential cognate receptor to the transc! ription complex via an alternative route.