Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Nairoviruses are an important group of tick-borne viruses that includes pathogens of man (Crimean Congo hemorrhagic fever virus) and livestock animals (Dugbe virus, Nairobi sheep disease virus (NSDV)). NSDV is found in large parts of East Africa and the Indian subcontinent (where it is known as Ganjam virus). We have investigated the ability of NSDV to antagonise the induction and actions of interferon. Both pathogenic and apathogenic isolates could actively inhibit the induction of type 1 interferon, and also blocked the signalling pathways of both type 1 and type 2 interferons. Using transient expression of viral proteins or sections of viral proteins, these activities all mapped to the ovarian tumour-like protease domain (OTU) found in the viral RNA polymerase. Virus infection, or expression of this OTU domain in transfected cells, led to a great reduction in the incorporation of ubiquitin or ISG15 protein into host cell proteins. Point mutations in the OTU that inhibited the protease activity also prevented it from antagonising interferon induction and action. Interestingly, a mutation at a peripheral site, which had little apparent effect on the ability of the OTU to inhibit ubiquitination and ISG15ylation, removed the ability of the OTU to block the induction of type 1 and the action of type 2 interferons, but had a lesser effect on the ability to block type 1 interferon action, suggesting that targets other than ubiquitin and ISG15 may be involved in the actions of the viral OTU.
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PMID:Inhibition of interferon induction and action by the nairovirus Nairobi sheep disease virus/Ganjam virus. 2216 42

The coordination of RNA polymerase I transcription with pre-rRNA processing, preribosomal particle assembly, and nuclear export is a finely tuned process requiring the concerted actions of a number of accessory factors. However, the exact functions of some of these proteins and how they assemble in subcomplexes remain poorly defined. LAS1L was first described as a nucleolar protein required for maturation of the 60S preribosomal subunit. In this paper, we demonstrate that LAS1L interacts with PELP1, TEX10, and WDR18, the mammalian homologues of the budding yeast Rix1 complex, along with NOL9 and SENP3, to form a novel nucleolar complex that cofractionates with the 60S preribosomal subunit. Depletion of LAS1L-associated proteins results in a p53-dependent G1 arrest and leads to defects in processing of the pre-rRNA internal transcribed spacer 2 region. We further show that the nucleolar localization of this complex requires active RNA polymerase I transcription and the small ubiquitin-like modifier-specific protease SENP3. Taken together, our data identify a novel mammalian complex required for 60S ribosomal subunit synthesis, providing further insight into the intricate, yet poorly described, process of ribosome biogenesis in higher eukaryotes.
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PMID:LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis. 2219 Jul 35

The production of mature and export competent mRNP (mRNA ribonucleoprotein) complexes depends on a series of highly coordinated processing reactions. RNA polymerase II (RNAPII) plays a central role in this process by mediating the sequential recruitment of mRNA maturation and export factors to transcribing genes, thereby establishing a strong functional link between transcription and export through nuclear pore complexes (NPC). Growing evidence indicates that post-translational modifications participate in the dynamic association of processing and export factors with mRNAs ensuring that the transitions and rearrangements undergone by the mRNP occur at the right time and place. This review mainly focuses on the role of ubiquitin conjugation in controlling mRNP assembly and quality control from transcription down to export through the NPC. It emphasizes the central role of ubiquitylation in organizing the chronology of events along this highly dynamic pathway. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
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PMID:Ubiquitin and assembly of export competent mRNP. 2224 Mar 87

The covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins, defined as sumoylation, is an important post-translational modification that regulates diverse cellular processes and many human diseases. However, functional analysis of sumo modification is usually hampered by the lack of sensitive methods for measuring extremely low abundance of specific sumoylated target in the cells. Here, we develop an ultrasensitive method for intracellular sumoylation assay based on SNAP tag (a mutant of O6-alkylguanine-DNA alkyltransferase)-mediated translation and RNA polymerase-based amplification. Intracellular sumo modification is first converted to the double-stranded DNA (dsDNA) containing the specific T7 promoter sequence via the covalent conjugation of SNAP tag with its substrate benzyl guanidine derivate; then, the dsDNA is extensively transcribed by T7 RNA polymerase to produce large amounts of RNAs, which are easily monitored using the RNA intercalating dye RiboGreen and a standard fluorometer. This method exhibits excellent specificity and high sensitivity and can detect as little as 5 pg of sumoylated p53 proteins, which has improved by as much as 1000-fold than that in the conventional Western blotting assay. Moreover, this method can measure intracellular sumoylation under different physiological conditions. Due to the common translation and amplification module, this method can be further extended to detect a variety of sumoylated proteins and other ubiquitin-like modifications in the cells and might provide a powerful tool for comprehensive analysis of the functions of sumoylation and other ubiquitin-like modifications in the fundamental biological processes and many human diseases.
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PMID:Sensitive detection of intracellular sumoylation via SNAP tag-mediated translation and RNA polymerase-based amplification. 2224 41

The small ubiquitin-related modifier (SUMO) is a conserved factor that post-translationally regulates proteins involved in many cellular processes, including gene transcription. We previously demonstrated that promoter-bound factors become sumoylated during activation of inducible genes in yeast, but the identity of these factors, and the role of sumoylation in their function, was unknown. Here we show that the transcriptional activator Gcn4 is sumoylated on two specific lysine residues and in a manner that depends on its ability to bind DNA, indicating that sumoylation occurs after Gcn4 binding to target promoters. Importantly, this functions to facilitate the subsequent removal of the activator from these promoters after recruitment of RNA polymerase II, which can prevent inappropriate transcription of target genes. Furthermore, we show that clearance of sumoylated Gcn4 requires the protein kinase and Mediator complex subunit Srb10, linking activator removal with target gene transcription. Our study demonstrates an unexpected role for protein sumoylation in the process of transcriptional activation.
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PMID:Sumoylation of transcription factor Gcn4 facilitates its Srb10-mediated clearance from promoters in yeast. 2234 16

Correct execution of mitosis in eukaryotes relies on timely activation and inactivation of cyclin B-dependent kinase 1 (cdk1), the M-phase-promoting factor (MPF). Once activated, MPF is sustained until mitotic spindle assembly by phosphorylation-dependent feedback loops that prevent inhibitory phosphorylation of cdk1 and ubiquitin-dependent degradation of cyclin B. Whether subsequent MPF inactivation and anaphase onset require a specific phosphatase(s) to reverse these feedback loops is not known. Here we show through biochemical and genetic evidence that timely MPF inactivation requires activity of the essential RNA polymerase II-carboxy-terminal domain phosphatase Fcp1, in a transcription-independent manner. We identify Cdc20, a coactivator of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) required for cyclin degradation and anaphase onset, USP44, a deubiquitinating peptidase that opposes APC/C action, and Wee1, a cdk1 inhibitory kinase, as relevant Fcp1 targets. We propose that Fcp1 has a crucial role in the liaison between dephosphorylation and ubiquitination that drives mitosis exit.
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PMID:Fcp1-dependent dephosphorylation is required for M-phase-promoting factor inactivation at mitosis exit. 2269 37

Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1) plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb), in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell morphologies, revealing a novel function of a conserved, co-transcriptional histone modification.
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PMID:A positive feedback loop links opposing functions of P-TEFb/Cdk9 and histone H2B ubiquitylation to regulate transcript elongation in fission yeast. 2287 98

Transcriptional activator-like (TAL) effectors of plant pathogenic bacteria function as transcription factors in plant cells. However, how TAL effectors control transcription in the host is presently unknown. Previously, we showed that TAL effectors of the citrus canker pathogen Xanthomonas citri, named PthAs, targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Here we show that CsCyp complements the function of Cpr1 and Ess1, two yeast cyclophilins that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD) of RNA polymerase II. We also demonstrate that CsCyp, CsTdx, CsUev and four PthA variants interact with the citrus CTD and that CsCyp co-immunoprecipitate with the CTD in citrus cell extracts and with PthA2 transiently expressed in sweet orange epicotyls. The interactions of CsCyp with the CTD and PthA2 were inhibited by cyclosporin A (CsA), a cyclophilin inhibitor. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase) activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in X. citri-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD.
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PMID:TAL effectors target the C-terminal domain of RNA polymerase II (CTD) by inhibiting the prolyl-isomerase activity of a CTD-associated cyclophilin. 2291 12

Ubiquitylation is a highly diverse and complex post-translational modification for the regulation of protein function and stability. Studies of ubiquitylation have, however, been hampered by its rapid reversal in cell extracts, for example through the action of de-ubiquitylating enzymes (DUBs). Here we describe a novel ubiquitin-binding protein reagent, MultiDsk, composed of an array of five UBA domains from the yeast ubiquitin-binding protein Dsk2, fused to GST. MultiDsk binds ubiquitylated substrates with unprecedented avidity, and can be used as both an affinity resin to study protein ubiquitylation, and to effectively protect ubiquitylated proteins from the action of DUBs and the proteasome in crude cell extracts. We use the resin to show that the Def1 protein becomes ubiquitylated in response to DNA damage, and to isolate ubiquitylated forms of RNA polymerase II.
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PMID:MultiDsk: a ubiquitin-specific affinity resin. 2305 98

Poxviruses are considered less dependent on host functions than other DNA viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for DNA and mRNA synthesis. Nevertheless, RNAi screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (VACV). Translational, ubiquitin-proteosome, and endoplasmic reticulum-to-Golgi transport functions, known to be important for VACV, were enriched in the siRNA-inhibiting group, and RNA polymerase II and associated functions were enriched in the siRNA-enhancing group. Additional findings, notably the inhibition of VACV spread by siRNAs to several nuclear pore genes, were unanticipated. Knockdown of nucleoporin 62 strongly inhibited viral morphogenesis, with only a modest effect on viral gene expression, recapitulating and providing insight into previous studies with enucleated cells.
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PMID:Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis. 2340 14


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