RESUMO
The Paf1 complex (Paf1C) affects RNA polymerase II transcription by coordinating co-transcriptional chromatin modifications and helping recruit mRNA 3' end processing factors. Paf1C cross-links to transcribed genes, but not downstream of the cleavage and polyadenylation site, suggesting that it may interact with the nascent mRNA. Paf1C purified from Saccharomyces cerevisiae binds RNA in vitro, as do the purified Leo1 and Rtf1 subunits of the complex. In vivo cross-linking and immunoprecipitation of RNA associated with Paf1C (RNA-IP) show that Leo1, but not Rtf1, is necessary for the complex to bind RNA. Cells lacking Leo1 have reduced Paf1C recruitment as well as decreased levels of histone H3 and trimethylated H3 Lys(4) within transcribed chromatin. Together, these results suggest that association of Paf1C with RNA stabilizes its localization at actively transcribed regions where it influences chromatin structure.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Histonas/química , Metilação , Proteínas Nucleares/química , Nucleossomos/metabolismo , Ligação Proteica , RNA/química , RNA/metabolismo , RNA Polimerase II/metabolismo , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation.
