scholarly journals Human Transcription Elongation Factor CA150 Localizes to Splicing Factor-Rich Nuclear Speckles and Assembles Transcription and Splicing Components into Complexes through Its Amino and Carboxyl Regions

2006 ◽  
Vol 26 (13) ◽  
pp. 4998-5014 ◽  
Author(s):  
Miguel Sánchez-Álvarez ◽  
Aaron C. Goldstrohm ◽  
Mariano A. Garcia-Blanco ◽  
Carlos Suñé
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Splicing Factor ◽  
Transcriptional Elongation ◽  
Nuclear Speckles ◽  
Ww Domains ◽  
Amino Terminal ◽  
Transcription Elongation Factor

ABSTRACT The human transcription elongation factor CA150 contains three N-terminal WW domains and six consecutive FF domains. WW and FF domains, versatile modules that mediate protein-protein interactions, are found in nuclear proteins involved in transcription and splicing. CA150 interacts with the splicing factor SF1 and with the phosphorylated C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) through its WW and FF domains, respectively. WW and FF domains may, therefore, serve to link transcription and splicing components and play a role in coupling transcription and splicing in vivo. In the study presented here, we investigated the subcellular localization and association of CA150 with factors involved in pre-mRNA transcriptional elongation and splicing. Endogenous CA150 colocalized with nuclear speckles, and this was not affected either by inhibition of cellular transcription or by RNAPII CTD phosphorylation. FF domains are essential for the colocalization to speckles, while WW domains are not required for colocalization. We also performed biochemical assays to understand the role of WW and FF domains in mediating the assembly of transcription and splicing components into higher-order complexes. Transcription and splicing components bound to a region in the amino-terminal part of CA150 that contains the three WW domains; however, we identified a region of the C-terminal FF domains that was also critical. Our results suggest that sequences located at both the amino and carboxyl regions of CA150 are required to assemble transcription/splicing complexes, which may be involved in the coupling of those processes.

scholarly journals The Transcription Elongation Factor CA150 Interacts with RNA Polymerase II and the Pre-mRNA Splicing Factor SF1

2001 ◽  
Vol 21 (22) ◽  
pp. 7617-7628 ◽  
Author(s):  
Aaron C. Goldstrohm ◽  
Todd R. Albrecht ◽  
Carles Suñé ◽  
Mark T. Bedford ◽  
Mariano A. Garcia-Blanco
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Nascent Transcript ◽  
Ww Domains ◽  
Transcription Elongation Factor ◽  
Carboxyl Terminal

ABSTRACT CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcripts. The FF repeat domains of CA150 bind directly to the phosphorylated carboxyl-terminal domain of the largest subunit of RNAPII. We determined that this interaction is required for efficient CA150-mediated repression of transcription from the α4-integrin promoter. Additional functional determinants, namely, the WW1 and WW2 domains of CA150, were also required for efficient repression. A protein that interacted directly with CA150 WW1 and WW2 was identified as the splicing-transcription factor SF1. Previous studies have demonstrated a role for SF1 in transcription repression, and we found that binding of the CA150 WW1 and WW2 domains to SF1 correlated exactly with the functional contribution of these domains for repression. The binding specificity of the CA150 WW domains was found to be unique in comparison to known classes of WW domains. Furthermore, the CA150 binding site, within the carboxyl-terminal half of SF1, contains a novel type of proline-rich motif that may be recognized by the CA150 WW1 and WW2 domains. These results support a model for the recruitment of CA150 to repress transcription elongation. In this model, CA150 binds to the phosphorylated CTD of elongating RNAPII and SF1 targets the nascent transcript.

scholarly journals Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

2009 ◽  
Vol 106 (17) ◽  
pp. 6956-6961 ◽  
Author(s):  
Karen Zhou ◽  
Wei Hung William Kuo ◽  
Jeffrey Fillingham ◽  
Jack F. Greenblatt
Keyword(s):  
Histone Modification ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Consensus Sequence ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Histone H2b ◽  
Paf Complex

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1–Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.

scholarly journals Runx1 Binds Positive Transcription Elongation Factor b and Represses Transcriptional Elongation by RNA Polymerase II: Possible Mechanism of CD4 Silencing

2005 ◽  
Vol 25 (24) ◽  
pp. 10675-10683 ◽  
Author(s):  
Huimin Jiang ◽  
Fan Zhang ◽  
Takeshi Kurosu ◽  
B. Matija Peterlin
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
T Cell Development ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Positive Transcription Elongation Factor ◽  
Transcription Elongation Factor ◽  
Inhibitory Domain

ABSTRACT Runx1 binds the silencer and represses CD4 transcription in immature thymocytes. In this study, we found that Runx1 inhibits P-TEFb, which contains CycT1, CycT2, or CycK and Cdk9 and stimulates transcriptional elongation by RNA polymerase II (RNAPII) in eukaryotic cells. Indeed, its inhibitory domain, spanning positions 371 to 411, not only bound CycT1 but was required for silencing CD4 transcription in vivo. Our chromatin immunoprecipitation assays revealed that Runx1 inhibits the elongation but not initiation of transcription and that RNAPII is engaged at the CD4 promoter but is unable to elongate in CD4− CD8+ thymoma cells. These results suggest that active repression by Runx1 occurs by blocking the elongation by RNAPII, which may contribute to CD4 silencing during T-cell development.

scholarly journals JMJD6 cleaves MePCE to release positive transcription elongation factor b (P-TEFb) in higher eukaryotes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Schuyler Lee ◽  
Haolin Liu ◽  
Ryan Hill ◽  
Chunjing Chen ◽  
Xia Hong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Factor B ◽  
Pol Ii ◽  
Positive Transcription Elongation Factor ◽  
Transcription Elongation Factor ◽  
Higher Eukaryotes

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex.

scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
Author(s):  
Junjiang Fu ◽  
Ho-Geun Yoon ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Complex Activity ◽  
Interacting Protein ◽  
Pol Ii ◽  
Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor

1990 ◽  
Vol 10 (10) ◽  
pp. 5433-5441
Author(s):  
B Y Ahn ◽  
P D Gershon ◽  
E V Jones ◽  
B Moss
Keyword(s):  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Viral Rna ◽  
In Vitro Translation ◽  
Virus Protein ◽  
Transcriptional Elongation

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface

2009 ◽  
Vol 425 (2) ◽  
pp. 373-380 ◽  
Author(s):  
Sabine Wenzel ◽  
Berta M. Martins ◽  
Paul Rösch ◽  
Birgitta M. Wöhrl
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Structural Similarity ◽  
Transcription Elongation Factor

The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity-inducing factor] is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In the present paper we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176–273) at a resolution of 1.55 Å (1 Å=0.1 nm). The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to the NTD (N-terminal domain) of the bacterial transcription factor NusG. A homologue for hSpt4 has not yet been found in bacteria. However, the archaeal transcription factor RpoE” appears to be distantly related. Although a comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4 could not be observed by NMR titration experiments. A conserved glutamate residue, which was shown to be crucial for dimerization in yeast, is also involved in the human heterodimer, but is substituted for a glutamine residue in Escherichia coli NusG. However, exchanging the glutamine for glutamate proved not to be sufficient to induce hSpt4 binding.

scholarly journals The Rpb6 Subunit of Fission Yeast RNA Polymerase II Is a Contact Target of the Transcription Elongation Factor TFIIS

2000 ◽  
Vol 20 (4) ◽  
pp. 1263-1270 ◽  
Author(s):  
Akira Ishiguro ◽  
Yasuhisa Nogi ◽  
Koji Hisatake ◽  
Masami Muramatsu ◽  
Akira Ishihama
Keyword(s):  
Rna Polymerase ◽  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Direct Interaction ◽  
Temperature Sensitive ◽  
Single Mutation ◽  
Transcription Elongation Factor ◽  
Essential Region

ABSTRACT The Rpb6 subunit of RNA polymerase II is one of the five subunits common to three forms of eukaryotic RNA polymerase. Deletion and truncation analyses of the rpb6 gene in the fission yeastSchizosaccharomyces pombe indicated that Rpb6, consisting of 142 amino acid residues, is an essential protein for cell viability, and the essential region is located in the C-terminal half between residues 61 and 139. After random mutagenesis, a total of 14 temperature-sensitive mutants were isolated, each carrying a single (or double in three cases and triple in one) mutation. Four mutants each carrying a single mutation in the essential region were sensitive to 6-azauracil (6AU), which inhibits transcription elongation by depleting the intracellular pool of GTP and UTP. Both 6AU sensitivity and temperature-sensitive phenotypes of these rpb6 mutants were suppressed by overexpression of TFIIS, a transcription elongation factor. In agreement with the genetic studies, the mutant RNA polymerases containing the mutant Rpb6 subunits showed reduced affinity for TFIIS, as measured by a pull-down assay of TFIIS-RNA polymerase II complexes using a fusion form of TFIIS with glutathioneS-transferase. Moreover, the direct interaction between TFIIS and RNA polymerase II was competed by the addition of Rpb6. Taken together, the results lead us to propose that Rpb6 plays a role in the interaction between RNA polymerase II and the transcription elongation factor TFIIS.

scholarly journals A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4445-4454 ◽  
Author(s):  
Dorothee Mueller ◽  
Christian Bach ◽  
Deniz Zeisig ◽  
Maria-Paz Garcia-Cuellar ◽  
Sara Monroe ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Modification ◽  
Histone H3 ◽  
Elongation Factor ◽  
Polycomb Group ◽  
Fusion Partner ◽  
Transcriptional Elongation ◽  
Associated Proteins ◽  
Group Members

Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79–specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.

Sign in / Sign up

Export Citation Format

Share Document