Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription

TLDR

mRNA processing factors are coupled to transcription through interactions with the highly phosphorylated carboxy‑terminal domain of RNA polymerase II, whose phosphorylation pattern changes during transcription and determines binding of enzymes such as the capping enzyme. The study aims to determine whether capping components remain associated with the transcription complex after they have modified the 5′ end of the mRNA. Chromatin immunoprecipitation in yeast shows that the capping enzyme binds only promoters, whereas the cap methyltransferase and Hrp1/CFIB bind both promoters and coding regions, and that Ser5 phosphorylation is promoter‑specific while Ser2 phosphorylation occurs in coding regions, indicating a dynamic association of processing factors with differently phosphorylated Pol II.

Abstract

The activities of several mRNA processing factors are coupled to transcription through binding to RNA polymerase II (Pol II). The largest subunit of Pol II contains a repetitive carboxy-terminal domain (CTD) that becomes highly phosphorylated during transcription. mRNA-capping enzyme binds only to phosphorylated CTD, whereas other processing factors may bind to both phosphorylated and unphosphorylated forms. Capping occurs soon after transcription initiation and before other processing events, raising the question of whether capping components remain associated with the transcription complex after they have modified the 5′ end of the mRNA. Chromatin immunoprecipitation in Saccharomyces cerevisiae shows that capping enzyme cross-links to promoters but not coding regions. In contrast, the mRNA cap methyltransferase and the Hrp1/CFIB polyadenylation factor cross-link to both promoter and coding regions. Remarkably, the phosphorylation pattern of the CTD changes during transcription. Ser 5 phosphorylation is detected primarily at promoter regions dependent on TFIIH. In contrast, Ser 2 phosphorylation is seen only in coding regions. These results suggest a dynamic association of mRNA processing factors with differently modified forms of the polymerase throughout the transcription cycle.