Serine-7 of the RNA Polymerase II CTD Is Specifically Required for snRNA Gene Expression

Abstract

RNA polymerase II (pol II) transcribes genes encoding proteins and non-coding small nuclear (sn)RNAs. The carboxy-terminal domain (CTD) of the largest subunit of mammalian RNA polymerase II (pol II), comprising tandem repeats of the heptapeptide consensus tyr1ser2pro3thr4ser5pro6ser7, is required for expression of both gene types. Here, we show that mutation of ser7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of ser7 facilitates interaction with the snRNA gene-specific Integrator complex. These findings asign 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.Human snRNA genes transcribed by pol II, including those encoding U1 and U2 spliceosomal RNAs, have specialized promoters comprising conserved proximal and distal sequence elements (PSE and DSE) (1). Rather than polyadenylation signals, 3′ box elements direct co-transcriptional formation of the primary 3′ end of transcripts (2, 3). The 3′ end of these pre-snRNAs is further processed in the cytoplasm to yields mature non-polyadenylated snRNAs (2). Removal of the CTD of the large subunit of mammalian pol II drastically affects expression of both snRNA and protein-coding genes (2-4). The CTD has a unique structure composed of multiple repeats containing residues that undergo reversible phosphorylation during transcription (5). For example, phosphorylation of ser5 by CDK7 facilitates promoter release and RNA capping, whereas ser2 phosphorylation by CDK9 is associated with processive elongation and 3′ processing (5,6). No role has yet been ascribed to ser7.The mammalian pol II CTD comprises 52 repeats, 25 of which deviate from the consensus at position 7. The mainly consensus repeats 1-25 activate snRNA 3′ processing more effectively than repeats 27-52, which have few serines at position 7 (2). In contrast, both halves of the CTD are equally effective in activating polyadenylation (7). We have tested the requirement for ser7 for expression of snRNA (U2G (2)) and mRNA (pCMV-hnRNPK (8)) templates in 293 cells by introducing mutations into consensus (Con) CTD repeats in an α-amanitin-resistant pol II large subunit (Rpb1) (9) ( Figures 1A, S1A). The large subunit of endogenous pol II is very sensitive to inhibition by α-amanitin, facilitating complementation studies (9). A CTD with at least 25 consensus repeats ((Con) 25 ) was used since this supports * To whom correspondence should be addressed. E-mail: shona.murphy@path.ox.ac.uk. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts efficient production and co-transcriptional 3′ processing of transcripts from snRNA and protein-coding templates, while five CTD repeats (Δ5) do not (2, 4) ( Figure S2A, B).Mutation of ser7 to the non-phospho-acceptor alanine (Ser7A) in a background of 25 repeats reduces the level of properly processed U2G transcripts (Proc) and increases the ratio of ...

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

“…A CTD sequence containing lysine at position 7 throughout was cloned following a similar strategy. GST-CTDs, designated as GST-CTD [9] or GST-CTD [13] according to the number of hepta-repeats, were expressed in BL21(DE3) or BL21 codon plus RP cells after induction with 0.1 mM isopropyl-β-d-thiogalactoside for 16 h at 20 °C. Protein purification was performed using GSH-affinity chromatography and subsequent size exclusion chromatography.…”

“…Protein purification was performed using GSH-affinity chromatography and subsequent size exclusion chromatography. In addition, a GST-CTD [8] construct was cloned with the N-and C-terminal three residues of the CTD [9] template mutated to lysines. This construct was designed for better ionization properties in the ESI-MS analyses of the otherwise fully negatively charged CTD protein.…”

“…Phosphorylation of Ser5 by Cdk7 and Cdk8 kinases of TFIIH and the mediator complex, respectively, has been described to be concomitant with transcription initiation, whereas Cdk9 of P-TEFb is suggested to phosphorylate Ser2, which marks the elongation phase of transcription [3][4][5][6][7][8] . In addition, Ser7 of the CTD can also be phosphorylated, which has been linked to small nuclear RNAs (snRNA) transcription and recruitment of the integrator complex [9][10][11][12][13] . Whereas some genes, for example, those on CpG islands, can directly proceed to the elongation state by recruiting P-TEFb to RNAPII 14 , genomewide studies suggest that the majority of genes in higher eukaryotes are under the control of promoter-proximal pausing [15][16][17] .…”

Transcription termination by nuclear RNA polymerases

“…However, it was recently demonstrated that mammalian RNAPII can also be phosphorylated at Ser7 of the CTD Egloff et al 2007). High levels of Ser7 phosphorylation were detected at promoter regions of protein-coding genes, with increasing levels toward the 39-region , as well as at spliceosomal snRNAs genes (Egloff et al 2007).…”

The multi-tasking P-TEFb complex

“…The RNAPII CTD is hypophosphorylated when initially recruited to genes, and undergoes sequential phosphorylation at Ser5 during promoter clearance and at Ser2 by P-TEFb (CycT1:Cdk9) at the start of elongation [4,5]. The CTD is also phosphorylated at the Ser7 position, which controls expression of snRNA genes [6,7]. In the absence of P-TEFb, Ser5P RNAPII complexes accumulate 20−40 nt downstream of the transcription start site, partly owing to the actions of the negative-acting elongation factor complex, NELF, and the DRB-sensitivity inducing complex, DSIF/Spt4,5 [8].…”