title: 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.
authors.affiliation: Ecole Normale Supérieure, IBENS, 46, rue d'Ulm, Paris 75005, France.
authors.forename: Bo
authors.initials: B
authors.lastname: Gu
authors.forename: Dirk
authors.initials: D
authors.lastname: Eick
authors.forename: Olivier
authors.initials: O
authors.lastname: Bensaude
mesh_terms: D000409:Alanine; D019943:Amino Acid Substitution; D002460:Cell Line; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D026721:RNA 3' End Processing; D059367:RNA Cleavage; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D012694:Serine / Q000502:physiology*; D000072668:Splicing Factor U2AF; D014158:Transcription, Genetic; D039221:mRNA Cleavage and Polyadenylation Factors / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; D021122:Protein Subunits; D016760:Proto-Oncogene Proteins c-fos; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C405483:U1 small nuclear RNA; C405484:U2 small nuclear RNA; C000607494:U2AF2 protein, human; D039221:mRNA Cleavage and Polyadenylation Factors; D012694:Serine; D012319:RNA Polymerase II; D000409:Alanine