Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression

Abstract

RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of serine-7 facilitates interaction with the snRNA gene-specific Integrator complex. These findings assign a biological function to this amino acid and highlight a gene type-specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.

Figure 1. Ser7 is required for expression of snRNA but not protein-coding templates

(A) RNase protection analysis of RNA transcribed from U2G or pCMV-hnRNPK constructs after ectopic expression of α-amanitin-resistant Rpb1 (see Figure S1A). (Con) designates consensus CTD heptapeptides. (B) RNase protection analysis of transcripts from endogenous U2 genes in cells stably expressing α-amanitin-resistant Rpb1 and Western blot analysis of Rpb1 expression. qRT-PCR analysis of U2 pre-snRNA and hElf-1 mRNA in total RNA normalized to 7SK RNA with α-amanitin-treated cells expressing no Rpb1 as negative control (NO POL). (C) Run-on analysis of endogenous U1 and U2 snRNA genes in cells transfected with α-amanitin-resistant Rpb1s. AS and Up are negative controls (see Materials and Methods). Quantitation of this data is shown in the bar graph below.

Figure 2. Mutation of ser7 to alanine affects association of Integrator with snRNA genes

(A) ChIP analysis of Rpb1 and TAP-Int9 associated with U1, U2, γ-actin and GAPDH promoters. (B) ChIP analysis of endogenous U2 genes using antibodies to Rpb1 (pol II) or phospho-serine7 (Ser7P). (C) Western blot analysis of GST-CTD “pull down” of Integrator using anti-Int11 antibodies (17). (D) Phosphorylation of ser7 is required for efficient interaction of Integrator with pol II. Disruption of this interaction may cause a defect in a post-recruitment step of transcription, in addition to affecting 3′ processing.