Role of the C-Terminal Domain of RNA Polymerase II in U2 snRNA Transcription and 3′ Processing

Abstract

U small nuclear RNAs (snRNAs) and mRNAs are both transcribed by RNA polymerase II (Pol II), but the snRNAs have unusual TATA-less promoters and are neither spliced nor polyadenylated; instead, 3′ processing is directed by a highly conserved 3′ end formation signal that requires initiation from an snRNA promoter. Here we show that the C-terminal domain (CTD) of Pol II is required for efficient U2 snRNA transcription, as it is for mRNA transcription. However, CTD kinase inhibitors, such as 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), that block mRNA elongation do not affect U2 transcription, although 3′ processing of the U2 primary transcript is impaired. We show further that U2 transcription is preferentially inhibited by low doses of UV irradiation or actinomycin D, which induce CTD kinase activity, and that UV inhibition can be rescued by treatment with DRB or H7. We propose that Pol II complexes transcribing snRNAs and mRNAs have distinct CTD phosphorylation patterns. mRNA promoters recruit factors including kinases that hyperphosphorylate the CTD, and the CTD in turn recruits proteins needed for mRNA splicing and polyadenylation. We predict that snRNA promoters recruit factors including a CTD kinase(s) whose snRNA-specific phosphorylation pattern recruits factors required for promoter-coupled 3′ end formation.

We thank Anton Krumm and Mark Groudine for advice on nuclear run-on transcription and Tom Pavelitz for assistance cloning pNE BS SK+. We also thank Arnold Bailey and John Newman for critical reading of the manuscript. The α-amanitin-resistant Pol II LS constructs were kind gifts of David Bentley (University of Colorado Health Sciences Center, Denver).

This work was supported by NIH GM41624 and NRSA T32 GM07270.