The Dynamic Association of RNA Polymerase II with Initiation, Elongation, and RNA Processing Factors during the Transcription Cycle

2007 ◽  
pp. 49-66
Author(s):  
Kristi L. Penheiter ◽  
Judith A. Jaehning
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Dynamic Association ◽  
Processing Factors ◽  
Transcription Cycle

scholarly journals Direct Interactions between the Paf1 Complex and a Cleavage and Polyadenylation Factor Are Revealed by Dissociation of Paf1 from RNA Polymerase II

2008 ◽  
Vol 7 (7) ◽  
pp. 1158-1167 ◽  
Author(s):  
Kristen Nordick ◽  
Matthew G. Hoffman ◽  
Joan L. Betz ◽  
Judith A. Jaehning
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Direct Interaction ◽  
Pol Ii ◽  
Phosphorylated Form ◽  
Cleavage And Polyadenylation ◽  
Paf1 Complex ◽  
Processing Factors ◽  
Polyadenylation Factor ◽  
Transcription Cycle

ABSTRACT The Paf1 complex (Paf1, Ctr9, Cdc73, Rtf1, and Leo1) is normally associated with RNA polymerase II (Pol II) throughout the transcription cycle. However, the loss of either Rtf1 or Cdc73 results in the detachment of the Paf1 complex from Pol II and the chromatin form of actively transcribed genes. Using functionally tagged forms of the Paf1 complex factors, we have determined that, except for the more loosely associated Rtf1, the remaining components stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin. The loss of Paf1, Ctr9, or to a lesser extent Cdc73 or Rtf1 results in reduced levels of serine 2 phosphorylation of the Pol II C-terminal domain and in increased read through of the MAK21 polyadenylation site. We found that the cleavage and polyadenylation factor Cft1 requires the Pol II-associated form of the Paf1 complex for full levels of interaction with the serine 5-phosphorylated form of Pol II. When the Paf1 complex is dissociated from Pol II, a direct interaction between Cft1 and the Paf1 complex can be detected. These results are consistent with the Paf1 complex providing a point of contact for recruitment of 3′-end processing factors at an early point in the transcription cycle. The lack of this connection helps to explain the defects in 3′-end formation observed in the absence of Paf1.

scholarly journals Human hHpr1/p84/Thoc1 Regulates Transcriptional Elongation and Physically Links RNA Polymerase II and RNA Processing Factors

2005 ◽  
Vol 25 (10) ◽  
pp. 4023-4033 ◽  
Author(s):  
Yanping Li ◽  
Xiaoling Wang ◽  
Xiaojing Zhang ◽  
David W. Goodrich
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Rna Splicing ◽  
Transcriptional Elongation ◽  
Intact Cells ◽  
Efficient Gene ◽  
Cellular Phenotypes ◽  
Processing Factors

ABSTRACT Cotranscriptional loading of RNA processing factors onto nascent RNA facilitates efficient gene expression. Mechanisms responsible for coupling transcription and RNA processing are not well defined, but the Saccharomyces cerevisiae TREX complex provides an example. TREX is composed of the subcomplex THO that associates with RNA polymerase II and is required for normal transcriptional elongation. THO associates with proteins involved in RNA splicing and export to form the larger TREX complex. Hence, assembly of TREX physically couples transcriptional elongation with RNA processing factors. Whether metazoan species with long, intron-containing genes utilize a similar mechanism has not been established. Here we show that human hHpr1/p84/Thoc1 associates with elongating RNA polymerase II and the RNA splicing and export factor UAP56 in intact cells. Depletion of hHpr1/p84/Thoc1 causes transcriptional elongation defects and associated cellular phenotypes similar to those observed in THO-deficient yeast. We conclude that hHpr1/p84/Thoc1 regulates transcriptional elongation and may participate in a protein complex functionally analogous to yeast TREX, physically linking elongating RNA polymerase II with RNA processing factors.

scholarly journals Coordination of transcription, processing, and export of highly expressed RNAs by distinct biomolecular condensates

2020 ◽  
Vol 4 (3) ◽  
pp. 281-291 ◽  
Author(s):  
Alexander M. Ishov ◽  
Aishwarya Gurumurthy ◽  
Jörg Bungert
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Transcription Initiation ◽  
Nuclear Speckles ◽  
High Concentration ◽  
Pol Ii ◽  
Processing Factors

Genes under control of super-enhancers are expressed at extremely high levels and are frequently associated with nuclear speckles. Recent data suggest that the high concentration of unphosphorylated RNA polymerase II (Pol II) and Mediator recruited to super-enhancers create phase-separated condensates. Transcription initiates within or at the surface of these phase-separated droplets and the phosphorylation of Pol II, associated with transcription initiation and elongation, dissociates Pol II from these domains leading to engagement with nuclear speckles, which are enriched with RNA processing factors. The transitioning of Pol II from transcription initiation domains to RNA processing domains effectively co-ordinates transcription and processing of highly expressed RNAs which are then rapidly exported into the cytoplasm.

scholarly journals An RNA Polymerase Pause Site Is Associated with the Immunoglobulin μs Poly(A) Site

2002 ◽  
Vol 22 (15) ◽  
pp. 5606-5615 ◽  
Author(s):  
Martha L. Peterson ◽  
Shannon Bertolino ◽  
Frankie Davis
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Immunoglobulin A ◽  
Expression Ratio ◽  
B Cell Maturation ◽  
Mrna Ratio ◽  
Site Use ◽  
A Site ◽  
Pause Site

ABSTRACT Immunoglobulin μ alternative RNA processing is regulated during B-cell maturation and requires balanced efficiencies of the competing splice (μm) and cleavage-polyadenylation (μs) reactions. When we deleted sequences 50 to 200 nucleotides beyond the μs poly(A) site, the μs/μm mRNA ratio decreased three- to eightfold in B, plasma, and nonlymphoid cells. The activity could not be localized to a smaller fragment but did function in heterologous contexts. Our data suggest that this region contains an RNA polymerase II pause site that enhances the use of the μs poly(A) site. First, known pause sites replaced the activity of the deleted fragment. Second, the μ fragment, when placed between tandem poly(A) sites, enhanced the use of the upstream poly(A) site. Finally, nuclear run-ons detected an increase in RNA polymerase loading just downstream from the μs poly(A) site, even when the poly(A) site was inactivated. When this μ fragment and another pause site were inserted 1 kb downstream from the μs poly(A) site, they no longer affected the mRNA expression ratio, suggesting that pause sites affect poly(A) site use over a limited distance. Fragments from the immunoglobulin A gene were also found to have RNA polymerase pause site activity.

scholarly journals Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation

2010 ◽  
Vol 30 (21) ◽  
pp. 5180-5193 ◽  
Author(s):  
Alicia García ◽  
Emanuel Rosonina ◽  
James L. Manley ◽  
Olga Calvo
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Terminal Domain ◽  
Mrna Metabolism ◽  
Genes Encoding ◽  
The One ◽  
Ctd Phosphorylation ◽  
Transcription Cycle

ABSTRACT The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3′-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

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