Separation of the Saccharomyces cerevisiae Paf1 Complex from RNA Polymerase II Results in Changes in Its Subnuclear Localization

ABSTRACT The yeast Paf1 complex (Paf1C), composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, associates with RNA polymerase II (Pol II) at promoters and in the actively transcribed portions of mRNA genes. Loss of Paf1 results in severe phenotypes and significantly reduced levels of the other Paf1C components. In contrast, loss of Rtf1 causes relatively subtle phenotypic changes and no reduction in the other Paf1C factors but disrupts the association of these factors with Pol II and chromatin. To elucidate the fate of the Paf1C when dissociated from Pol II, we examined the localization of the Paf1C components in paf1 and rtf1 mutant yeast strains. We found that although the Paf1C factors remain nuclear in paf1 and rtf1 strains, loss of Paf1 or Rtf1 results in a change in the subnuclear distribution of the remaining factors. In wild-type cells, Paf1C components are present in the nucleoplasm but not the nucleolus. In contrast, in both paf1 and rtf1 strains, the remaining factors are found in the nucleolus as well as the nucleoplasm. Loss of Paf1 affects nucleolar function; we observed that expression of MAK21 and RRP12, important for rRNA processing, is reduced concomitant with an increase in rRNA precursors in a paf1 strain. However, these changes are not the result of relocalization of the Paf1C because loss of Rtf1 does not cause similar changes in rRNA processing. Instead, we speculate that the change in localization may reflect a link between the Paf1C and newly synthesized mRNAs as they exit the nucleus.

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Ctr9, Rtf1, and Leo1 Are Components of the Paf1/RNA Polymerase II Complex

Molecular and Cellular Biology ◽

10.1128/mcb.22.7.1971-1980.2002 ◽

2002 ◽

Vol 22 (7) ◽

pp. 1971-1980 ◽

Cited By ~ 144

Author(s):

Cherie L. Mueller ◽

Judith A. Jaehning

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Double Mutant ◽

Affinity Purification ◽

Tata Binding Protein ◽

Temperature Sensitive ◽

Wild Type ◽

Pol Ii ◽

Paf1 Complex ◽

Transcriptional Start Sites

ABSTRACT The Saccharomyces cerevisiae Paf1-RNA polymerase II (Pol II) complex is biochemically and functionally distinct from the Srb-mediator form of Pol II holoenzyme and is required for full expression of a subset of genes. In this work we have used tandem affinity purification tags to isolate the Paf1 complex and mass spectrometry to identify additional components. We have established that Ctr9, Rtf1, and Leo1 are factors that associate with Paf1, Cdc73, and Pol II, but not with the Srb-mediator. Deletion of either PAF1 or CTR9 leads to similar severe pleiotropic phenotypes, which are unaltered when the two mutations are combined. In contrast, we found that deletion of LEO1 or RTF1 leads to few obvious phenotypes, although mutation of RTF1 suppresses mutations in TATA-binding protein, alters transcriptional start sites, and affects elongation. Remarkably, deletion of LEO1 or RTF1 suppresses many paf1Δ phenotypes. In particular, an rtf1Δ paf1Δ double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1Δ single mutant. In addition, expression of the G1 cyclin CLN1, reduced nearly threefold in paf1Δ, is restored to wild-type levels in the rtf1Δ paf1Δ double mutant. We suggest that lack of Paf1 results in a defective complex and a block in transcription, which is relieved by removal of Leo1 or Rtf1.

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Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

Journal of Biological Chemistry ◽

10.1074/jbc.m009634200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 12266-12273 ◽

Cited By ~ 31

Author(s):

Wenxiang Wei ◽

Dorjbal Dorjsuren ◽

Yong Lin ◽

Weiping Qin ◽

Takahiro Nomura ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Middle Part ◽

Wild Type ◽

Binding Region ◽

Pol Ii ◽

Transcription Factor Iif ◽

B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

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Rpb7 Can Interact with RNA Polymerase II and Support Transcription during Some Stresses Independently of Rpb4

Molecular and Cellular Biology ◽

10.1128/mcb.19.4.2672 ◽

1999 ◽

Vol 19 (4) ◽

pp. 2672-2680 ◽

Cited By ~ 48

Author(s):

Ayelet Sheffer ◽

Mazal Varon ◽

Mordechai Choder

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Stress Conditions ◽

Cold Sensitivity ◽

Starvation Period ◽

Wild Type ◽

Pol Ii ◽

Growth Phenotype ◽

Mild Temperature

ABSTRACT Rpb4 and Rpb7 are two yeast RNA polymerase II (Pol II) subunits whose mechanistic roles have recently started to be deciphered. Although previous data suggest that Rpb7 can stably interact with Pol II only as a heterodimer with Rpb4, RPB7 is essential for viability, whereas RPB4 is essential only during some stress conditions. To resolve this discrepancy and to gain a better understanding of the mode of action of Rpb4, we took advantage of the inability of cells lacking RPB4 (rpb4Δ, containing Pol IIΔ4) to grow above 30°C and screened for genes whose overexpression could suppress this defect. We thus discovered that overexpression of RPB7 could suppress the inability ofrpb4Δ cells to grow at 34°C (a relatively mild temperature stress) but not at higher temperatures. Overexpression ofRPB7 could also partially suppress the cold sensitivity ofrpb4Δ strains and fully suppress their inability to survive a long starvation period (stationary phase). Notably, however, overexpression of RPB4 could not override the requirement for RPB7. Consistent with the growth phenotype, overexpression of RPB7 could suppress the transcriptional defect characteristic of rpb4Δ cells during the mild, but not during a more severe, heat shock. We also demonstrated, through two reciprocal coimmunoprecipitation experiments, a stable interaction of the overproduced Rpb7 with Pol IIΔ4. Nevertheless, fewer Rpb7 molecules interacted with Pol IIΔ4 than with wild-type Pol II. Thus, a major role of Rpb4 is to augment the interaction of Rpb7 with Pol II. We suggest that Pol IIΔ4 contains a small amount of Rpb7 that is sufficient to support transcription only under nonstress conditions. When RPB7 is overexpressed, more Rpb7 assembles with Pol IIΔ4, enough to permit appropriate transcription also under some stress conditions.

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The Spt4p Subunit of Yeast DSIF Stimulates Association of the Paf1 Complex with Elongating RNA Polymerase II

Molecular and Cellular Biology ◽

10.1128/mcb.26.8.3135-3148.2006 ◽

2006 ◽

Vol 26 (8) ◽

pp. 3135-3148 ◽

Cited By ~ 54

Author(s):

Hongfang Qiu ◽

Cuihua Hu ◽

Chi-Ming Wong ◽

Alan G. Hinnebusch

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Histone Methylation ◽

Histone H3 ◽

Coding Sequences ◽

Pol Ii ◽

Cell Extracts ◽

Carboxy Terminal Domain ◽

Paf1 Complex ◽

Carboxy Terminal

ABSTRACT The Paf1 complex (Paf1C) interacts with RNA polymerase II (Pol II) and promotes histone methylation of transcribed coding sequences, but the mechanism of Paf1C recruitment is unknown. We show that Paf1C is not recruited directly by the activator Gcn4p but is dependent on preinitiation complex assembly and Ser5 carboxy-terminal domain phosphorylation for optimal association with ARG1 coding sequences. Importantly, Spt4p is required for Paf1C occupancy at ARG1 (and other genes) and for Paf1C association with Ser5-phosphorylated Pol II in cell extracts, whereas Spt4p-Pol II association is independent of Paf1C. Since spt4Δ does not reduce levels of Pol II at ARG1, Ser5 phosphorylation, or Paf1C expression, it appears that Spt4p (or its partner in DSIF, Spt5p) provides a platform on Pol II for recruiting Paf1C following Ser5 phosphorylation and promoter clearance. spt4Δ reduces trimethylation of Lys4 on histone H3, demonstrating a new role for yeast DSIF in promoting a Paf1C-dependent function in elongation.

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The Transcriptional Intermediary Factor TIF1γ Controls Erythroid Cell Fate by Specifically Regulating Transcriptional Elongation.

Blood ◽

10.1182/blood.v114.22.254.254 ◽

2009 ◽

Vol 114 (22) ◽

pp. 254-254

Author(s):

Xiaoying Bai ◽

Joseph Lee ◽

Jocelyn LeBlanc ◽

Anna Sessa ◽

Zhongan Yang ◽

...

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cell Fate ◽

Erythroid Cell ◽

Transcriptional Elongation ◽

Pol Ii ◽

Physical Interactions ◽

Paf1 Complex

Abstract Abstract 254 Vertebrate erythropoiesis is regulated by cell-specific transcription factors, RNA polymerase-associated basal machinery and chromatin remodeling factors. One critical chromatin factor is the transcriptional intermediary factor TIF1γ. Loss of TIF1γfunction in zebrafish mutant moonshine causes a profound anemia during embryogenesis, associated with a progressive decrease in expression of most erythroid mRNAs such as GATA1 and globin. TIF1γdeficiency has also been linked to TGF-βsignaling, although the in vivo mechanism for the anemia remains unclear. In an effort to find genes that interact with TIF1γ, we undertook a genetic suppressor screen in which we sought mutations in another gene that would restore blood to normal levels in the background of moonshine deficiency. Few suppressor screens have been done in vertebrate genetic models, and the haploid genetics of zebrafish was a great advantage for this screen. After screening 800 families of fish, two suppressor mutants, “eclipse” and “sunrise”, were found that could greatly rescue the erythroid defects in moonshine. The deficient gene in sunrise has been mapped to the locus of cdc73 (also known as parafibromin/HRPT2), a subunit of the PAF1 complex known to regulate RNA polymerase II (Pol II) elongation and chromatin modification. Furthermore, we have found that knocking down other subunits in the PAF1 complex also rescued the blood defect in moonshine, suggesting that PAF1 as a complex antagonizes TIF1γfunction during erythropoiesis. In yeast, PAF1 has been shown to physically or genetically interact with other elongation factors including DSIF, FACT and p-TEFb. We have found that knocking down DSIF, which is known to induce Pol II pausing during early elongation, also rescues moonshine. FACT and p-TEFb are both known to counteract DSIF to release Pol II from pausing, and knocking down FACT and p-TEFb caused the zebrafish to develop anemia. This strongly suggests that the erythroid defects in TIF1γdeficiency is caused by attenuated Pol II elongation. In an effort to understand the cell-specific phenotype of TIF1γdeficiency, we introduced a FLAG tagged TIF1γinto K562 erythroleukemia cells to pull down interacting proteins. Physical interactions were found among TIF1γ, FACT, p-TEFb and surprisingly the SCL hematopoietic transcription complex. The interaction with the SCL complex provides a cell-specific control over transcriptional elongation. The physical interactions, taken together with the genetic data, suggest a novel mechanism regulating erythropoiesis. TIF1γphysically and functionally links blood-specific transcription factors like SCL to Pol II-associated elongation machinery to regulate blood cell fate. In light of the recent discoveries of widespread Pol II stalling in the promoter proximal region in metazoan genomes, we speculate that similar mechanisms will regulate cell fates in other blood lineages as well as non-blood tissues. Disclosures: Zon: FATE Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Stemgent: Consultancy.

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

Eukaryotic Cell ◽

10.1128/ec.00434-07 ◽

2008 ◽

Vol 7 (7) ◽

pp. 1158-1167 ◽

Cited By ~ 58

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.

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Histone H2B Ubiquitylation Is Associated with Elongating RNA Polymerase II

Molecular and Cellular Biology ◽

10.1128/mcb.25.2.637-651.2005 ◽

2005 ◽

Vol 25 (2) ◽

pp. 637-651 ◽

Cited By ~ 221

Author(s):

Tiaojiang Xiao ◽

Cheng-Fu Kao ◽

Nevan J. Krogan ◽

Zu-Wen Sun ◽

Jack F. Greenblatt ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Transcription Elongation ◽

Lysine Methylation ◽

Coding Region ◽

Elongation Complex ◽

Histone H2b ◽

Pol Ii ◽

Paf1 Complex

ABSTRACT Rad6-mediated ubiquitylation of histone H2B at lysine 123 has been linked to transcriptional activation and the regulation of lysine methylation on histone H3. However, how Rad6 and H2B ubiquitylation contribute to the transcription and histone methylation processes is poorly understood. Here, we show that the Paf1 transcription elongation complex and the E3 ligase for Rad6, Bre1, mediate an association of Rad6 with the hyperphosphorylated (elongating) form of RNA polymerase II (Pol II). This association appears to be necessary for the transcriptional activities of Rad6, as deletion of various Paf1 complex members or Bre1 abolishes H2B ubiquitylation (ubH2B) and reduces the recruitment of Rad6 to the promoters and transcribed regions of active genes. Using the inducible GAL1 gene as a model, we find that the recruitment of Rad6 upon activation occurs rapidly and transiently across the gene and coincides precisely with the appearance of Pol II. Significantly, during GAL1 activation in an rtf1 deletion mutant, Rad6 accumulates at the promoter but is absent from the transcribed region. This fact suggests that Rad6 is recruited to promoters independently of the Paf1 complex but then requires this complex for entrance into the coding region of genes in a Pol II-associated manner. In support of a role for Rad6-dependent H2B ubiquitylation in transcription elongation, we find that ubH2B levels are dramatically reduced in strains bearing mutations of the Pol II C-terminal domain (CTD) and abolished by inactivation of Kin28, the serine 5 CTD kinase that promotes the transition from initiation to elongation. Furthermore, synthetic genetic array analysis reveals that the Rad6 complex interacts genetically with a number of known or suspected transcription elongation factors. Finally, we show that Saccharomyces cerevisiae mutants bearing defects in the pathway to H2B ubiquitylation display transcription elongation defects as assayed by 6-azauracil sensitivity. Collectively, our results indicate a role for Rad6 and H2B ubiquitylation during the elongation cycle of transcription and suggest a mechanism by which H3 methylation may be regulated.

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Numerous post-translational modifications of RNA polymerase II subunit Rpb4 link transcription to post-transcriptional mechanisms

10.1101/798603 ◽

2019 ◽

Author(s):

Stephen Richard ◽

Lital Gross ◽

Jonathan Fischer ◽

Keren Bendalak ◽

Tamar Ziv ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

The Other ◽

Mrna Synthesis ◽

Mutant Type ◽

Post Translational Modifications ◽

Pol Ii ◽

One Stage

AbstractRpb4/7 binds RNA Polymerase II (Pol II) transcripts co-transcriptionally and accompanies them throughout their lives. By virtue of its capacity to interact with key regulators (e.g., Pol II, eIF3, Pat1) both temporarily and spatially, Rpb4/7 regulates the major stages of the mRNA lifecycle. Here we show that Rpb4/7 can undergo over 100 combinations of post-translational modifications (PTMs). Remarkably, the Rpb4/7 PTMs repertoire changes as the mRNA/Rpb4/7 complex progresses from one stage to the next. A mutagenesis approach in residues that undergo PTMs suggests that temporal Rpb4 PTMs regulate its interactions with key regulators of gene expression that control transcriptional and post-transcriptional stages. Moreover, one mutant type specifically affects mRNA synthesis despite its normal association with Pol II, whereas the other affects both mRNA synthesis and decay; both types disrupt the balance between mRNA synthesis and decay (‘mRNA buffering’) and the cell’s capacity to respond to the environment. Taken together, we propose that temporal Rpb4/7 PTMs are involved in cross talks among the various stages of the mRNA lifecycle.

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Interplay between consensus and divergent RNA polymerase II C-terminal domain repeats in viability and targeting

10.1101/448506 ◽

2018 ◽

Author(s):

Feiyue Lu ◽

Bede Portz ◽

David S. Gilmour

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Signal Sequence ◽

Repetitive Sequences ◽

Wild Type ◽

Pol Ii ◽

Sequence Complexity ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Carboxy Terminal

SummaryThe carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) is composed of repeats of the consensus YSPTSPS, and is an essential binding scaffold for transcription-associated factors. Metazoan CTDs have well-conserved lengths and sequence compositions arising from the evolution of divergent motifs, features thought to be essential for development. To the contrary, we show that a truncated CTD composed solely of YSPTSPS repeats fully supports Drosophila viability, but a CTD with enough YSPTSPS repeats to match the length of the wild-type Drosophila CTD is defective. Furthermore, a fluorescently-tagged CTD lacking the rest of Pol II dynamically enters transcription compartments, indicating that the CTD functions as a signal sequence. However, CTDs with too many YSPTSPS repeats are more prone to localize to static nuclear foci independent of the chromosomes. We propose that the sequence complexity of the CTD offsets aberrant behavior caused by excessive repetitive sequences without compromising its targeting function.

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The FACT Complex Travels with Elongating RNA Polymerase II and Is Important for the Fidelity of Transcriptional Initiation In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8323-8333.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8323-8333 ◽

Cited By ~ 237

Author(s):

Paul B. Mason ◽

Kevin Struhl

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Tata Binding Protein ◽

Dependent Manner ◽

Wild Type ◽

Transcriptional Initiation ◽

Pol Ii ◽

Coding Regions

ABSTRACT The FACT complex facilitates transcription on chromatin templates in vitro, and it has been functionally linked to nucleosomes and putative RNA polymerase II (Pol II) elongation factors. In Saccharomyces cerevisiae cells, FACT specifically associates with active Pol II genes in a TFIIH-dependent manner and travels across the gene with elongating Pol II. Conditional inactivation of the FACT subunit Spt16 results in increased Pol II density, transcription, and TATA-binding protein (TBP) occupancy in the 3′ portion of certain coding regions, indicating that FACT suppresses inappropriate initiation from cryptic promoters within coding regions. Conversely, loss of Spt16 activity reduces the association of TBP, TFIIB, and Pol II with normal promoters. Thus, FACT is required for wild-type cells to restrict initiation to normal promoters, thereby ensuring that only appropriate mRNAs are synthesized. We suggest that FACT contributes to the fidelity of Pol II transcription by linking the processes of initiation and elongation.

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