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CTD serine-2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo.

ABSTRACT: Co-transcriptional pre-mRNA processing relies on reversible phosphorylation of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNAP II). In this study, we replaced in live cells the endogenous Rpb1 by S2A Rpb1, where the second serines (Ser2) in the CTD heptapeptide repeats were switched to alanines, to prevent phosphorylation. Although slower, S2A RNAP II was able to transcribe. However, it failed to recruit splicing components such as U2AF65 and U2 snRNA to transcription sites, although the recruitment of U1 snRNA was not affected. As a consequence, co-transcriptional splicing was impaired. Interestingly, the magnitude of the S2A RNAP II splicing defect was promoter dependent. In addition, S2A RNAP II showed an impaired recruitment of the cleavage factor PCF11 to pre-mRNA and a defect in 3'-end RNA cleavage. These results suggest that CTD Ser2 plays critical roles in co-transcriptional pre-mRNA maturation in vivo: It likely recruits U2AF65 to ensure an efficient co-transcriptional splicing and facilitates the recruitment of pre-mRNA 3'-end processing factors to enhance 3'-end cleavage.

SUBMITTER: Gu B

PROVIDER: S-EPMC3561981 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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CTD serine-2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo.

Gu Bo B Eick Dirk D Bensaude Olivier O

Nucleic acids research 20121228 3

Co-transcriptional pre-mRNA processing relies on reversible phosphorylation of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNAP II). In this study, we replaced in live cells the endogenous Rpb1 by S2A Rpb1, where the second serines (Ser2) in the CTD heptapeptide repeats were switched to alanines, to prevent phosphorylation. Although slower, S2A RNAP II was able to transcribe. However, it failed to recruit splicing components such as U2AF65 and U2 snRNA t ...[more]

PMID: 23275552

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Project description:Transcriptional activity of RNA polymerase II (Pol II) is influenced by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes simplex virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) by targeting CDK9 and other CDKs, but the full functional implications of this are not well understood. Using Western blotting, we report that HSV-1 also induces a loss of serine 7 phosphorylation (pS7) of the CTD during lytic infection, requiring expression of the two immediate-early proteins ICP22 and ICP27. ICP27 has also been proposed to target RPB1 for degradation, but we show that pS2/S7 loss precedes the drop in total protein levels. Cells with the RPB1 polyubiquitination site mutation K1268R, preventing proteasomal degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained higher overall RPB1 protein levels later in infection, indicating this pathway is not involved in early CTD dysregulation but may mediate bulk protein loss later. Using α-amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for the production of viral proteins, with Ser2 facilitating viral immediate-early genes and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments with immunofluorescence. These data expand the known means that HSV employs to create pro-viral transcriptional environments at the expense of host responses.IMPORTANCECells rapidly induce changes in the transcription of RNA in response to stress and pathogens. Herpes simplex virus (HSV) disrupts many processes of host mRNA transcription, and it is necessary to separate the actions of viral proteins from cellular responses. Here, we demonstrate that viral proteins inhibit two key phosphorylation patterns on the C-terminal domain (CTD) of cellular RNA polymerase II and that this is separate from the degradation of polymerases later in infection. Furthermore, we show that viral genes do not require the full "CTD code." Together, these data distinguish multiple steps in the remodeling of RNA polymerase during infection and suggest that shared transcriptional phenotypes during stress responses do not revolve around a core disruption of CTD modifications.

Dataset Information

CTD serine-2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo.

Publications

CTD serine-2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo.

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