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)

An RNA-binding protein gene (rbp1) from Drosophila melanogaster, encoding an RNA recognition motif and an Arg-Ser rich (RS) domain, has been characterized. The predicted amino acid sequence of rbp1 is similar to those of the human splicing factor ASF/SF2, the Drosophila nuclear phosphoprotein SRp55, and the Drosophila puff-associated protein B52. Northern and immunohistochemical analyses showed that rbp1 is expressed at all stages in all tissues and that the RBP1 protein is localized to the nucleus. Consistent with a role in mRNA metabolism, indirect immunofluorescence reveals that the RBP1 protein colocalizes with RNA polymerase II on larval salivary gland polytene chromosomes. RBP1 protein made in Escherichia coli was tested for splicing activity using human cell extracts in which ASF has been shown previously both to activate splicing and to affect the choice of splice sites in alternatively spliced pre-mRNAs. In these assays, RBP1 protein, like ASF, is capable of both activating splicing and switching splice site selection. However, in each case, clear differences in the behavior of the two proteins were detected, suggesting that they have related but not identical functions. The general nuclear expression pattern, colocalization on chromosomes with RNA polymerase II, the similarity to ASF/SF2, SRp55, and B52, along with the effect on alternative splicing shown in vitro, suggest that rbp1 is involved in the processing of precursor mRNAs.
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PMID:The Drosophila RNA-binding protein RBP1 is localized to transcriptionally active sites of chromosomes and shows a functional similarity to human splicing factor ASF/SF2. 134 Apr 70

Drosophila B52 protein is a homologue of human ASF/SF2 that functions in vitro as an essential pre-mRNA splicing factor. Immunofluorescence analysis of polytene chromosomes has shown that B52 generally colocalizes with RNA polymerase II; however, in contrast to other splicing factors, B52 brackets RNA polymerase II at highly active heat-shock puffs. Also, UV cross-linking in nonpolytene cells has shown that B52 cross-links in vivo to DNA flanking the highly active transcription units. Here, we find that the distribution of cross-linked B52 at heat-shock loci depends on transcription levels. Heat shocks at low and moderate temperatures, which induce corresponding levels of transcription, recruit B52 both to transcribed DNA and to flanking DNA, whereas a full heat-shock induction concentrates B52 on the DNA that brackets the entire activated region. We have also identified a 46-kDa protein from Chironomus tentans that binds Drosophila B52 antibodies and has a distribution on chromosomes analogous to B52. This protein is found throughout the moderately transcribed Balbiani rings. However, when transcription at these rings is hyperinduced to levels comparable to fully induced Drosophila heat-shock genes, the protein is restricted to the boundaries of highly decondensed chromatin. We suggest that B52 tracks to chromatin fibers that are folding or unfolding, and we discuss this in light of B52's proposed roles in pre-mRNA splicing and control.
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PMID:Distribution of B52 within a chromosomal locus depends on the level of transcription. 818 67

SR protein ASF/SF2 is a general pre-mRNA splicing factor as well as a regulator of alternative splicing. Data presented here show that ASF/SF2 is efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. At the intermediate stages of a productive infection, ASF/SF2 colocalizes with small nuclear ribonucleoprotein particles (snRNPs), splicing factors in ring-like structures surrounding viral replication centres and, at late stages of the infection, in enlarged speckles. Results presented here demonstrate that ASF/SF2 requires only one of the two RNA-recognition motifs (RRMs) present in the protein for its efficient recruitment to the ring-like structures, where viral pre-mRNAs are transcribed and processed, and that the arginine/serine-rich (RS) domain in ASF/SF2 is both redundant and insufficient for the translocation of the protein to active viral RNA polymerase II genes in adenovirus-infected cells.
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PMID:A single RNA recognition motif in splicing factor ASF/SF2 directs it to nuclear sites of adenovirus transcription. 1499 43

RNA polymerase II, and specifically the C-terminal domain (CTD) of its largest subunit, has been demonstrated to play important roles in capping, splicing, and 3' processing of mRNA precursors. But how the CTD functions in these reactions, especially splicing, is not well understood. To address some of the basic questions concerning CTD function in splicing, we constructed and purified two fusion proteins, a protein in which the CTD is positioned at the C terminus of the splicing factor ASF/SF2 (ASF-CTD) and an RS domain deletion mutant protein (ASFDeltaRS-CTD). Significantly, compared to ASF/SF2, ASF-CTD increased the reaction rate during the early stages of splicing, detected as a 20- to 60-min decrease in splicing lag time depending on the pre-mRNA substrate. The increased splicing rate correlated with enhanced production of prespliceosomal complex A and the early spliceosomal complex B but, interestingly, not the very early ATP-independent complex E. Additional assays indicate that the RS domain and CTD perform distinct functions, as exemplified by our identification of an activity that cooperates only with the CTD. Dephosphorylated ASFDeltaRS-CTD and a glutathione S-transferase-CTD fusion protein were both inactive, suggesting that an RNA-targeting domain and CTD phosphorylation were necessary. Our results provide new insights into the mechanism by which the CTD functions in splicing.
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PMID:The C-terminal domain of RNA polymerase II functions as a phosphorylation-dependent splicing activator in a heterologous protein. 1563 56

SR proteins constitute a family of pre-mRNA splicing factors now thought to play several roles in mRNA metabolism in metazoan cells. Here we provide evidence that a prototypical SR protein, ASF/SF2, is unexpectedly required for maintenance of genomic stability. We first show that in vivo depletion of ASF/SF2 results in a hypermutation phenotype likely due to DNA rearrangements, reflected in the rapid appearance of DNA double-strand breaks and high-molecular-weight DNA fragments. Analysis of DNA from ASF/SF2-depleted cells revealed that the nontemplate strand of a transcribed gene was single stranded due to formation of an RNA:DNA hybrid, R loop structure. Stable overexpression of RNase H suppressed the DNA-fragmentation and hypermutation phenotypes. Indicative of a direct role, ASF/SF2 prevented R loop formation in a reconstituted in vitro transcription reaction. Our results support a model by which recruitment of ASF/SF2 to nascent transcripts by RNA polymerase II prevents formation of mutagenic R loop structures.
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PMID:Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. 1609 57

The cellular protein p32 is a multifunctional protein, which has been shown to interact with a large number of cellular and viral proteins and to regulate several important activities like transcription and RNA splicing. We have previously shown that p32 regulates RNA splicing by binding and inhibiting the essential SR protein ASF/SF2. To determine whether p32 also functions as a regulator of splicing in virus-infected cells, we constructed a recombinant adenovirus expressing p32 under the transcriptional control of an inducible promoter. Much to our surprise the results showed that p32 overexpression effectively blocked mRNA and protein expression from the adenovirus major late transcription unit (MLTU). Interestingly, the p32-mediated inhibition of MLTU transcription was accompanied by an approximately 4.5-fold increase in Ser 5 phosphorylation and an approximately 2-fold increase in Ser 2 phosphorylation of the carboxy-terminal domain (CTD). Further, in p32-overexpressing cells the efficiency of RNA polymerase elongation was reduced approximately twofold, resulting in a decrease in the number of polymerase molecules that reached the end of the major late L1 transcription unit. We further show that p32 stimulates CTD phosphorylation in vitro. The inhibitory effect of p32 on MLTU transcription appears to require the CAAT box element in the major late promoter, suggesting that p32 may become tethered to the MLTU via an interaction with the CAAT box binding transcription factor.
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PMID:Cellular splicing and transcription regulatory protein p32 represses adenovirus major late transcription and causes hyperphosphorylation of RNA polymerase II. 1664 Dec 92

Cap formation is the first step of pre-mRNA processing in eukaryotic cells. Immediately after transcription initiation, capping enzyme (CE) is recruited to RNA polymerase II (Pol II) by the phosphorylated carboxyl-terminal domain of the Pol II largest subunit (CTD), allowing cotranscriptional capping of the nascent pre-mRNA. Recent studies have indicated that CE affects transcription elongation and have suggested a checkpoint model in which cotranscriptional capping is a necessary step for the early phase of transcription. To investigate further the role of the CTD in linking transcription and processing, we generated a fusion protein of the mouse CTD with T7 RNA polymerase (CTD-T7 RNAP). Unexpectedly, in vitro transcription assays with CTD-T7 RNAP showed that CE promotes formation of DNA.RNA hybrids or R loops. Significantly, phosphorylation of the CTD was required for CE-dependent R-loop formation (RLF), consistent with a critical role for the CTD in CE recruitment to the transcription complex. The guanylyltransferase domain was necessary and sufficient for RLF, but catalytic activity was not required. In vitro assays with appropriate synthetic substrates indicate that CE can promote RLF independent of transcription. ASF/SF2, a splicing factor known to prevent RLF, and GTP, which affects CE conformation, antagonized CE-dependent RLF. Our findings suggest that CE can play a direct role in transcription by modulating displacement of nascent RNA during transcription.
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PMID:Human capping enzyme promotes formation of transcriptional R loops in vitro. 1797 74

The human immunodeficiency virus type 1 (HIV-1) Tat is a 14-kDa viral protein that acts as a potent transactivator by binding to the transactivation-responsive region, a structured RNA element located at the 5' end of all HIV-1 transcripts. Tat transactivates viral gene expression by inducing the phosphorylation of the C-terminal domain of RNA polymerase II through several Tat-activated kinases and by recruiting chromatin-remodeling complexes and histone-modifying enzymes to the HIV-1 long terminal repeat. Histone acetyltransferases, including p300 and hGCN5, not only acetylate histones but also acetylate Tat at lysine positions 50 and 51 in the arginine-rich motif. Acetylated Tat at positions 50 and 51 interacts with a specialized protein module, the bromodomain, and recruits novel factors having this particular domain, such as P/CAF and SWI/SNF. In addition to having its effect on transcription, Tat has been shown to be involved in splicing. In this study, we demonstrate that Tat interacts with cyclin-dependent kinase 13 (CDK13) both in vivo and in vitro. We also found that CDK13 increases HIV-1 mRNA splicing and favors the production of the doubly spliced protein Nef. In addition, we demonstrate that CDK13 acts as a possible restriction factor, in that its overexpression decreases the production of the viral proteins Gag and Env and subsequently suppresses virus production. Using small interfering RNA against CDK13, we show that silencing of CDK13 leads to a significant increase in virus production. Finally, we demonstrate that CDK13 mediates its effect on splicing through the phosphorylation of ASF/SF2.
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PMID:CDK13, a new potential human immunodeficiency virus type 1 inhibitory factor regulating viral mRNA splicing. 1848 Apr 52

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate expression of their target messenger RNAs. We recently demonstrated that primary miRNA transcripts (pri-miRNAs) retained at transcription sites are processed with enhanced efficiency, suggesting that pri-miRNA processing is coupled to transcription in mammalian cells. We also observed that transiently expressed pri-miRNAs accumulate in nuclear foci with splicing factor SC35 and Microprocessor components, Drosha and DGCR8. Here, we show that pri-miRNAs containing a self-cleaving hepatitis delta ribozyme accumulate in the nucleoplasm after release from their transcription sites, but are not efficiently processed. Pri-miRNAs with ribozyme-generated 3' ends do not localize to SC35-containing foci, whereas cleaved and polyadenylated pri-miRNA transcripts with or without the pre-miRNA hairpin do. Pri-miRNA/SC35 foci contain a number of proteins normally associated with SC35 domains, including ASF/SF2, PABII, and the prolyl isomerase, Pin1. In contrast, RNA polymerase II and PM/Scl-100 do not strongly colocalize with pri-miRNAs in SC35-containing foci. These data argue that pri-miRNA/SC35-containing foci are not major sites of pri-miRNA processing and that pri-miRNA processing is coupled to transcription. We discuss the implications of our findings relative to recent insights into miRNA biogenesis, mRNA metabolism, and the nuclear organization of gene expression.
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PMID:Subnuclear compartmentalization of transiently expressed polyadenylated pri-microRNAs: processing at transcription sites or accumulation in SC35 foci. 1917 9

DNA-damaging agents cause a multifaceted cellular stress response. Cells set in motion either repair mechanisms or programmed cell death pathways, depending on the extent of the damage and on their ability to withstand it. The RNA-binding protein (RBP) Sam68, which is up-regulated in prostate carcinoma, promotes prostate cancer cell survival to genotoxic stress. Herein, we have investigated the function of Sam68 in this cellular response. Mitoxantrone (MTX), a topoisomerase II inhibitor, induced relocalization of Sam68 from the nucleoplasm to nuclear granules, together with several other RBPs involved in alternative splicing, such as TIA-1, hnRNP A1 and the SR proteins SC35 and ASF/SF2. Sam68 accumulation in nuclear stress granules was independent of signal transduction pathways activated by DNA damage. Using BrU labelling and immunofluorescence, we demonstrate that MTX-induced nuclear stress granules are transcriptionally active foci where Sam68 and the phosphorylated form of RNA polymerase II accumulate. Finally, we show that MTX-induced relocalization of Sam68 correlates with changes in alternative splicing of its mRNA target CD44, and that MTX-induced CD44 splicing depends on Sam68 expression. These results strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell that modulates alternative splicing in response to DNA damage.
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PMID:Genotoxic stress causes the accumulation of the splicing regulator Sam68 in nuclear foci of transcriptionally active chromatin. 2011 Feb 58


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