RNA Polymerase II Carboxy-Terminal Domain Phosphorylation Is Required for Cotranscriptional Pre-mRNA Splicing and 3′-End Formation

Abstract

We investigated the role of RNA polymerase II (pol II) carboxy-terminal domain (CTD) phosphorylation in pre-mRNA processing coupled and uncoupled from transcription in Xenopus oocytes. Inhibition of CTD phosphorylation by the kinase inhibitors 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole and H8 blocked transcription-coupled splicing and poly(A) site cleavage. These experiments suggest that pol II CTD phosphorylation is required for efficient pre-mRNA splicing and 3′-end formation in vivo. In contrast, processing of injected pre-mRNA was unaffected by either kinase inhibitors or α-amanitin-induced depletion of pol II. pol II therefore does not appear to participate directly in posttranscriptional processing, at least in frog oocytes. Together these experiments show that the influence of the phosphorylated CTD on pre-mRNA splicing and 3′-end processing is mediated by transcriptional coupling.

We thank T. Blumenthal and J. Jaehning for helpful discussions. We are grateful to the UCHSC Cancer Center sequencing facility, the UCHSC Center for Laboratory Animal Care, and to N. Fong for anti-CstF p77.

This work was supported by NIH grant GM58163 to D.B., a predoctoral training grant in molecular biology (NIH T32-GM0870) to G.B., and a Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation fellowship (DRG-1626) to D.A.R.Z.