The Rpb4/7 Module of RNA Polymerase II Is Required for Carbon Catabolite Repressor Protein 4-Negative on TATA (Ccr4-Not) Complex to Promote Elongation

Babbarwal, Vinod; Fu, Jiyang; Reese, Joseph C.

doi:10.1074/jbc.c114.601088

Cited by 37 publications

(56 citation statements)

References 26 publications

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“…At this point, we propose that Asr1 ubiquitylates Rpb1-and likely other factors-the result of which is ejection of the Rpb4/7 heterodimer and termination of subtelomeric transcription. Although Rpb4/7 are dispensable for transcriptional elongation in vitro (6), this model is compatible with our observation that Asr1-associated pol II is catalytically inactive (5) and that transcriptional elongation by pol II in vivo can be stimulated by Rpb4/7 (17)(18)(19). After transcription is terminated, we suggest that corecruitment of Ubp3 with Asr1 allows rapid reversal of Rpb1 ubiquitylation, reassembly of the pol II complex, and restoration of polymerase function.…”

Section: Discussionsupporting

confidence: 87%

Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

McCann¹,

Guo²,

Tansey³

2016

Proc. Natl. Acad. Sci. U.S.A.

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Ubiquitin, and components of the ubiquitin-proteasome system, feature extensively in the regulation of gene transcription. Although there are many examples of how ubiquitin controls the activity of transcriptional regulators and coregulators, there are few examples of core components of the transcriptional machinery that are directly controlled by ubiquitin-dependent processes. The budding yeast protein Asr1 is the prototypical member of the RPC (RING, PHD, CBD) family of ubiquitin-ligases, characterized by the presence of aminoterminal RING (really interesting new gene) and PHD (plant homeo domain) fingers and a carboxyl-terminal domain that directly binds the largest subunit of RNA polymerase II (pol II), Rpb1, in response to phosphorylation events tied to the initiation of transcription. Asr1-mediated oligo-ubiquitylation of pol II leads to ejection of two core subunits of the enzyme and is associated with inhibition of polymerase function. Here, we present evidence that Asr1-mediated ubiquitylation of pol II is required for silencing of subtelomeric gene transcription. We show that Asr1 associates with telomere-proximal chromatin and that disruption of the ubiquitin-ligase activity of Asr1-or mutation of ubiquitylation sites within Rpb1-induces transcription of silenced gene sequences. In addition, we report that Asr1 associates with the Ubp3 deubiquitylase and that Asr1 and Ubp3 play antagonistic roles in setting transcription levels from silenced genes. We suggest that control of pol II by nonproteolytic ubiquitylation provides a mechanism to enforce silencing by transient and reversible inhibition of pol II activity at subtelomeric chromatin.ubiquitin | chromatin | gene silencing | transcription T he ubiquitin (Ub)-proteasome system features in a multitude of cellular processes, including the regulation of transcription (1). Over the past two decades, it has become clear that Ubdependent processes impact multiple steps in gene expression, ranging from signaling histone modifications (2) to coordinating events of mRNA export from the nucleus (3). Many of the functions of Ub in transcription are directed against regulatory factors, and there are only a handful of examples in which core components of the transcriptional machinery are regulated by Ub-dependent transactions. The best understood example from this category is the Ub-mediated destruction of the largest subunit of RNA polymerase II (pol II) in response to DNA damage (4), which functions as a failsafe mechanism to remove stalled pol II complexes from damaged DNA segments. Beyond a damage response, however, it is unclear whether ubiquitylation controls the normal activity of pol II.We previously reported that the Saccharomyces cerevisiae protein Asr1 is a RING (really interesting new gene) finger Ub-ligase that targets RNA polymerase II for nonproteolytic ubiquitylation (5). Asr1 defines the evolutionarily preserved RPC (RING, PHD, CBD) family of Ub-ligases, characterized by the presence of amino-terminal RING and PHD (plant homeo domain) fingers an...

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Section: Discussionsupporting

confidence: 87%

Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

McCann¹,

Guo²,

Tansey³

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Additionally, the sensitivity of the transcript to single-stranded specific RNase indicates that extended RNA-DNA hybrids were not formed on the vast majority of the templates. All of the data described here, and elsewhere (28,(43)(44)(45)(46), indicate that this template produces ECs with the expected nucleic acid scaffold.…”

Section: Characterization Of Elongation Complexes Formed From Endinitmentioning

confidence: 99%

“…Templates-We use a minimal system for assembling ECs from highly purified components (28,(43)(44)(45)(46). RNAPII elongation was initiated from a 3Ј single strand extension on a duplex template to assemble an EC with a defined transcript produced from a G-less cassette (Fig.…”

Section: Characterization Of Elongation Complexes Formed From Endinitmentioning

confidence: 99%

Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest

Crickard

Reese

2016

Journal of Biological Chemistry

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RNA polymerase II (RNAPII) undergoes structural changes during the transitions from initiation, elongation, and termination, which are aided by a collection of proteins called elongation factors. NusG/Spt5 is the only elongation factor conserved in all domains of life. Although much information exists about the interactions between NusG/Spt5 and RNA polymerase in prokaryotes, little is known about how the binding of eukaryotic Spt4/5 affects the biochemical activities of RNAPII. We characterized the activities of Spt4/5 and interrogated the structural features of Spt5 required for it to interact with elongation complexes, bind nucleic acids, and promote transcription elongation. The eukaryotic specific regions of Spt5 containing the Kyrpides, Ouzounis, Woese domains are involved in stabilizing the association with the RNAPII elongation complex, which also requires the presence of the nascent transcript. Interestingly, we identify a region within the conserved NusG N-terminal (NGN) domain of Spt5 that contacts the non-template strand of DNA both upstream of RNAPII and in the transcription bubble. Mutating charged residues in this region of Spt5 did not prevent Spt4/5 binding to elongation complexes, but abrogated the cross-linking of Spt5 to DNA and the anti-arrest properties of Spt4/5, thus suggesting that contact between Spt5 (NGN) and DNA is required for Spt4/5 to promote elongation. We propose that the mechanism of how Spt5/NGN promotes elongation is fundamentally conserved; however, the eukaryotic specific regions of the protein evolved so that it can serve as a platform for other elongation factors and maintain its association with RNAPII as it navigates genomes packaged into chromatin.The conversion of DNA to RNA is a fundamental aspect of all life, and this process is carried out by RNA polymerases (RNAPs).2 These enzymatic powerhouses must maintain both high levels of fidelity and processivity over long distances to ensure that RNAs are accurately produced on a time scale amenable to life. Families of proteins called elongation factors have evolved to assist RNA polymerases during transcription elongation. The oldest and most conserved of these factors is the NusG/suppressor of Ty element (Spt) 5 family (1, 2). NusG is the eubacterial version of Spt5 and functions as a single polypeptide; however, archaea and eukaryotic Spt5 associate with an additional small protein, Spt4. In yeast, SPT5 is essential, but SPT4 is not. Deleting the gene encoding Spt4 impairs elongation, transcription-coupled repair, and mRNA processing (2-5). Some of the functions of Spt4 may be partially dependent on its ability to prevent degradation of Spt5 in cells (4). The NusG/Spt5 family of proteins has been shown to enhance RNA polymerase transcription elongation in all domains of life (6 -10). NusG regulates RNAP activity by stabilizing the post-translocated state thereby inhibiting backtracking and reducing long lifetime pauses (6, 11). The NusG homolog RfaH has also been implicated in regulating movement of the RNAP bridge hel...

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“…The Ccr4-Not complex was purified from 16-liter cultures of yeast containing a tandem affinity purification (TAP)-tagged version of CAF40 and a deletion of DST1, as described in previous publications (24,25). The purified complex was dialyzed in dialysis buffer (20 mM HEPES, pH 7.6, 150 mM KCl, 10% glycerol), followed by concentration using a microfiltration device.…”

Section: Methodsmentioning

confidence: 99%

“…Though the possibility that it has the ability to affect the elongation stage of transcription has been suggested by genetic studies (23), only recently have biochemical analysis confirmed that it plays a direct role in stimulating transcription elongation (22,24). The Ccr4-Not complex binds to elongation complexes (ECs) by directly interacting with both the Rpb4/Rpb7 module of RNAPII and the emerging transcript (25). Once it is bound to arrested ECs, it reactivates backtracked RNAPII, which apparently does not involve transcript cleavage.…”

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confidence: 99%

Ccr4-Not and TFIIS Function Cooperatively To Rescue Arrested RNA Polymerase II

Dutta

Babbarwal

et al. 2015

Molecular and Cellular Biology

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Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies in Saccharomyces cerevisiae yeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription.T ranscription of genes by RNA polymerase II (RNAPII) is a well-orchestrated process that involves steps of initiation, elongation, and termination. Following promoter clearance, RNAPII enters the phase of productive elongation that is achieved by a Brownian ratchet mechanism, in which the RNAPII oscillates between a pretranslocated and a posttranslocated state. After nucleotide addition to the 3= end of the RNA, the incoming nucleoside triphosphate (NTP) locks RNAPII in a posttranslocated form, readying it for the next cycle (1-4). However, productive elongation is not a product of efficient addition of nucleotides by RNAPII alone. During transcription elongation, RNAPII encounters several blocks, including sequence-specific pause sites, nucleotide limitations, DNA lesions, negative elongation factors, and DNA-bound proteins, which cause RNAPII to pause, arrest, or terminate transcription (5, 6). A myriad of elongation factors helps rescue paused/arrested polymerases and stimulate transcription (7,8). Each elongation factor acts via a different mechanism, and often, one works in combination with others.One of the best-characterized elongation factors known to rescue backtracked RNAPII is TFIIS. TFIIS promotes transcription elongation by stimulating the nucleolytic activity of RNAPII and realigning the 3= end of the transcript in the active site of arrested RNAPII (for reviews, see references 9 and 10). New evidence suggests that the cleavage-promoting activity of TFIIS is not the only way in which it stimulates elongation (11-13). Biochemical and biophysical studies have only recently begun to uncover the mechanisms by which TFIIS functions with TFII...

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The Rpb4/7 Module of RNA Polymerase II Is Required for Carbon Catabolite Repressor Protein 4-Negative on TATA (Ccr4-Not) Complex to Promote Elongation

Cited by 37 publications

References 26 publications

Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest

Ccr4-Not and TFIIS Function Cooperatively To Rescue Arrested RNA Polymerase II

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