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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 C-terminal domain (CTD) of the large subunit of RNA polymerase (pol) II comprises conserved heptad repeats, and post-translational modification of the CTD regulates transcription and cotranscriptional RNA processing. Recently, the spatial patterns of modification of the CTD repeats have been investigated, and new functions of CTD modification have been revealed. In addition, there are new insights into the roles of the enzymes that decorate the CTD. We review these new findings and reassess the role of the pol II CTD in the regulation of gene expression.

The positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1/T2, stimulates transcription by phosphorylating RNA polymerase II. The 7SK small nuclear RNA, in cooperation with HEXIM1 protein, functions as a general polymerase II transcription regulator by sequestering P-TEFb into a large kinase-inactive 7SK/HEXIM1/P-TEFb complex. Here, determination and characterization of the functionally essential elements of human 7SK snRNA directing HEXIM1 and P-TEFb binding led to a new model for the assembly of the 7SK/HEXIM1/P-TEFb regulatory complex. We demonstrate that two structurally and functionally distinct protein binding elements located in the 5-and 3-terminal hairpins of 7SK support the in vivo recruitment of HEXIM1 and P-TEFb. Consistently, a minimal regulatory RNA composed of the 5 and 3 hairpins of 7SK can modulate polymerase II transcription in HeLa cells. HEXIM1 binds independently and specifically to the G24-C48/G60-C87 distal segment of the 5 hairpin of 7SK. Binding of HEXIM1 is a prerequisite for association of P-TEFb with the G302-C324 apical region of the 3 hairpin of 7SK that is highly reminiscent of the human immunodeficiency virus transactivation-responsive RNA.Cyclic phosphorylation of the tandemly repeated YSPTSPS heptapeptide motif in the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is crucial for stimulating mRNA production (9, 14). For example, during the early stage of transcription, phosporylation of the CTD at serine 2 by the positive transcription elongation factor b (PTEFb) is essential for the transition from abortive to productive transcription elongation (5,7,30,35). P-TEFb is a general transcription factor that facilitates the production of fulllength mRNAs of most, if not all, protein-coding genes and also stimulates the Pol II-mediated synthesis of human immunodeficiency virus (HIV) transcripts from the 5Ј long terminal repeat of the integrated proviral genome (5, 29). P-TEFb is composed of a cyclin-dependent kinase, Cdk9, and the regulatory subunit cyclin (Cyc) T1, T2, or K (22, 33, 41; reviewed in reference 23). In human HeLa cells, about half of P-TEFb is associated with large ribonucleoprotein (RNP) complexes which also contain the 7SK small nuclear RNA (snRNA) and the HEXIM1 or HEXIM2 protein (3,17,20,36,37,39). In contrast to its free form, the 7SK/HEXIM1-associated fraction of P-TEFb shows little CTD kinase activity, indicating that the 7SK snRNA, in collaboration with HEXIM1, functions as an inhibitory factor of P-TEFb. Association of P-TEFb with 7SK/HEXIM1 is specific and reversible. Inhibition of transcription by chemical or UV treatment induces dissociation of P-TEFb from the kinase-inactive 7SK/ HEXIM1/P-TEFb complex (17,20,36,37). Consequently, increased accumulation of free P-TEFb facilitates CTD phosphorylation and mRNA production. Likewise, depletion of 7SK snRNA increases the CTD kinase activity of P-TEFb and stimulates transcription from Pol II-specific promoters, including the HIV long terminal repeat...