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 ...

The human snRNA genes transcribed by RNA polymerase II (e.g. U1 and U2) have a characteristic TATA-less promoter containing an essential proximal sequence element. Formation of the 3¢ end of these non-polyadenylated RNAs requires a specialized 3¢ box element whose function is promoter speci®c. Here we show that truncation of the C-terminal domain (CTD) of RNA polymerase II and treatment of cells with CTD kinase inhibitors, including DRB (5,6-dichloro-1-b-D-ribofuranosylbenzimidazole), causes a dramatic reduction in proper 3¢ end formation of U2 transcripts. Activation of 3¢ box recognition by the phosphorylated CTD would be consistent with the role of phospho-CTD in mRNA processing. CTD kinase inhibitors, however, have little effect on initiation or elongation of transcription of the U2 genes, whereas elongation of transcription of the b-actin gene is severely affected. This result highlights differences in transcription of snRNA and mRNA genes.

Phosphorylation of Ser2 of the heptapeptide repeat of the CTD of mammalian pol II by P-TEFb is associated with productive elongation of transcription of protein-coding genes. Here, we show that the CTD of pol II transcribing the human U2 snRNA genes is phosphorylated on Ser2 in vivo and that both the CDK9 kinase and cyclin T components of P-TEFb are required for cotranscriptional recognition of the 3 0 box RNA 3 0 end processing signal. However, inhibitors of CDK9 do not affect transcription of the U2 genes, indicating that P-TEFb functions exclusively as an RNA processing factor in expression of these relatively short, intronless genes. We also show that inhibition of CDK9 does not adversely affect either transcription of an intron-less, replication-activated histone H2b gene or recognition of the histone gene-specific U7-dependent RNA 3 0 end formation signal. These results emphasize that the role of P-TEFb as an activator of transcription elongation can be separated from its role in RNA processing and that neither function is universally required for expression of mammalian pol II-dependent genes.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) comprises multiple tandem repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. This unusual structure serves as a platform for the binding of factors required for expression of pol II-transcribed genes, including the small nuclear RNA (snRNA) gene-specific Integrator complex. The pol II CTD specifically mediates recruitment of Integrator to the promoter of snRNA genes to activate transcription and direct 3′ end processing of the transcripts. Phosphorylation of the CTD and a serine in position 7 are necessary for Integrator recruitment. Here, we have further investigated the requirement of the serines in the CTD heptapeptide and their phosphorylation for Integrator binding. We show that both Ser2 and Ser7 of the CTD are required and that phosphorylation of these residues is necessary and sufficient for efficient binding. Using synthetic phosphopeptides, we have determined the pattern of the minimal Ser2/Ser7 double phosphorylation mark required for Integrator to interact with the CTD. This novel double phosphorylation mark is a new addition to the functional repertoire of the CTD code and may be a specific signal for snRNA gene expression.