Ser7 Phosphorylation of the CTD Recruits the RPAP2 Ser5 Phosphatase to snRNA Genes

Egloff, Sylvain; Zaborowska, Justyna; Laitem, Clélia; Kiss, Tamás; Murphy, Shona

doi:10.1016/j.molcel.2011.11.006

Cited by 119 publications

(168 citation statements)

References 39 publications

(91 reference statements)

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“…In this study, we show that S. cerevisiae Rtr1 is a regulator of Ser5 phosphorylation in the RNAPII CTD in agreement with previous findings (14,39,41,49,51,52). We show that deletion of RTR1 leads to global increases in Ser5-P CTD occupancy at RNAPII target genes, suggesting that Rtr1 is a general regulator of RNAPII phosphorylation during transcription elongation.…”

supporting

confidence: 92%

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation

Hunter

Fox

Smith-Kinnaman

et al. 2016

Molecular and Cellular Biology

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cIn eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y 1 S 2 P 3 T 4 S 5 P 6 S 7 ) n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1. Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes. R eversible phosphorylation of the C-terminal domain (CTD)of the largest subunit of RNA polymerase II (RNAPII) controls transcription elongation and termination through the orderly recruitment of transcription regulatory factors. These factors are involved in various processes including mRNA processing and cotranscriptional histone modifications (1-3). The CTD is comprised of the heptapeptide repeat of Y 1 S 2 P 3 T 4 S 5 P 6 S 7 that is highly conserved with 26 repeats in Saccharomyces cerevisiae, 42 repeats in Drosophila melanogaster, and 52 repeats in humans and is essential for viability (4-10). The CTD is an unstructured domain that is unique to RNAPII and contains some degenerative repeats that deviate from the canonical sequence (11). Multiple residues within the CTD repeats are phosphorylated (P) at different stages of the transcription cycle; these residues include Ser2, Ser5, Ser7, Tyr1, and Tyr4, which thereby serve as a signaling scaffold for RNAPII transcription machinery (12). The phosphorylation of Ser2, Ser5, and Ser7 in S. cerevisiae has been well characterized by several laboratories (13,14). Phosphorylation of Tyr1 and Thr4 are less characterized but have also been implicated in the regulation of RNAPII transcription (12, 15). Additionally, the proline at position six in the CTD repeats is subject to cis-trans isomerization by Ess1 (16,17). Despite a great deal of research into these modifications, the extent to which these modifications coexist in a phosphorylated and nonphosphorylated state on a single heptapeptide repeat in vivo remains enigmatic.Th...

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supporting

confidence: 92%

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation

Hunter

Fox

Smith-Kinnaman

et al. 2016

Molecular and Cellular Biology

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“…Depletion of any of these factors would be predicted to extinguish U7-GFP promoter activity and would not result in GFP expression. Interestingly, we also did not score the fly ortholog of RPAP2 (CG34183), which has been suggested to play an important role in mediating Integrator's association to the RNAPII CTD (Egloff et al 2012). Depleting fly RPAP2 did not give rise to snRNA misprocessing in our hands, suggesting that RPAP2 may fall into the group of factors we mentioned above or may not be critical in Drosophila.…”

Section: Discussionmentioning

confidence: 63%

An RNAi screen identifies additional members of the Drosophila Integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3′-end formation

Chen¹,

Ezzeddine²,

Waltenspiel

et al. 2012

RNA

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Formation of the 39 end of RNA polymerase II-transcribed snRNAs requires a poorly understood group of proteins called the Integrator complex. Here we used a fluorescence-based read-through reporter that expresses GFP in response to snRNA misprocessing and performed a genome-wide RNAi screen in Drosophila S2 cells to identify novel factors required for snRNA 39-end formation. In addition to the known Integrator complex members, we identified Asunder and CG4785 as additional Integrator subunits. Functional and biochemical experiments revealed that Asunder and CG4785 are additional core members of the Integrator complex. We also identified a conserved requirement in both fly and human snRNA 39-end processing for cyclin C and Cdk8 that is distinct from their function in the Mediator Cdk8 module. Moreover, we observed biochemical association between Integrator proteins and cyclin C/Cdk8, and that overexpression of a kinase-dead Cdk8 causes snRNA misprocessing. These data functionally define the Drosophila Integrator complex and demonstrate an additional function for cyclin C/Cdk8 unrelated to its function in Mediator.

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“…Alternatively, stringent Ser5 dephosphorylation by phosphatases such as Rtr1 or Fcp1 in yeast 43,44 is required in order to render Ser2 phosphorylation possible. A recent study has indeed identified that the human RPAP2 phosphatase specifically recognizes Ser7 phosphorylation marks on the CTD to facilitate Ser5 dephosphorylation 45 . In a similar line of argument, a universal RNAPII CTD cycle along genes has been proposed, which is orchestrated by complex interplays between kinases, phosphatases and proline isomerases 46 .…”

Section: Y S P T S P S Y S P T S P S Y S P T S P Smentioning

confidence: 99%

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

2012

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Phosphorylation of RnA polymerase II carboxy-terminal domain (CTD) in hepta-repeats YsPTsPs regulates eukaryotic transcription. Whereas ser5 is phosphorylated in the initiation phase, ser2 phosphorylation marks the elongation state. Here we show that the positive transcription elongation factor P-TEFb is a ser5 CTD kinase that is unable to create ser2/ser5 double phosphorylations, while it exhibits fourfold higher activity on a CTD substrate prephosphorylated at ser7 compared with the consensus hepta-repeat or the YsPTsPK variant. mass spectrometry reveals an equal number of phosphorylations to the number of heptarepeats provided, yet the mechanism of phosphorylation is distributive despite the repetitive nature of the substrate. Inhibition of P-TEFb activity is mediated by two regions in Hexim1 that act synergistically on Cdk9 and Cyclin T1. HIV-1 Tat/TAR abrogates Hexim1 inhibition to stimulate transcription of viral genes but does not change the substrate specificity. Together, these results provide insight into the multifaceted pattern of CTD phosphorylation.

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Ser7 Phosphorylation of the CTD Recruits the RPAP2 Ser5 Phosphatase to snRNA Genes

Cited by 119 publications

References 39 publications

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation

An RNAi screen identifies additional members of the Drosophila Integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3′-end formation

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

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