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 herpes simplex virus type 1 tegument protein VP22 is known to be highly phosphorylated during infection. Here we show that two electrophoretic forms of VP22 can be identified in infected cell extracts and that this heterogeneity is accounted for by phosphorylation. Furthermore, the nonphosphorylated form of VP22 appears to be specifically incorporated into virions. We also show that the phosphorylated form of VP22 is the only form detected during transient transfection and as such that VP22 can act as a substrate for a cellular kinase. Phospho-amino acid and phospho-peptide analyses of in vivo labeled VP22 were utilized to demonstrate that the phosphorylation profiles of VP22 synthesized during transfection and infection are the same. In both cases VP22 was modified solely on serine residues located in the N-terminal 120 residues of the protein. Moreover, in vitro phosphorylation was utilized to show that the constitutive cellular kinase, casein kinase II, which has four serine consensus recognition sites at the N-terminus of VP22, phosphorylates VP22 in the same manner as observed in vivo. This kinase also phosphorylates VP22 at the N-terminus in intact capsid-tegument structures. Casein kinase II is therefore likely to be the major kinase of VP22 during infection.

Human U1 small nuclear (sn)RNA, required for splicing of pre-mRNA, is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes, also located on chromosome 1 (1q12-21), were thought to be pseudogenes. However, many of these “variant” (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells, we have identified global transcriptome changes following interrogation of the Affymetrix Human Exon ST 1.0 array. Our results indicate that this vU1 snRNA regulates expression of a subset of target genes at the level of pre-mRNA processing. This is the first indication that variant U1 snRNAs have a biological function in vivo. Furthermore, some vU1 snRNAs are packaged into unique ribonucleoproteins (RNPs), and many vU1 snRNA genes are differentially expressed in human embryonic stem cells (hESCs) and HeLa cells, suggesting developmental control of RNA processing through expression of different sets of vU1 snRNPs.

In addition to protein-coding genes, mammalian pol II (RNA polymerase II) transcribes independent genes for some non-coding RNAs, including the spliceosomal U1 and U2 snRNAs (small nuclear RNAs). snRNA genes differ from protein-coding genes in several key respects and some of the mechanisms involved in expression are gene-type-specific. For example, snRNA gene promoters contain an essential PSE (proximal sequence element) unique to these genes, the RNA-encoding regions contain no introns, elongation of transcription is P-TEFb (positive transcription elongation factor b)-independent and RNA 3'-end formation is directed by a 3'-box rather than a cleavage and polyadenylation signal. However, the CTD (C-terminal domain) of pol II closely couples transcription with RNA 5' and 3' processing in expression of both gene types. Recently, it was shown that snRNA promoter-specific recognition of the 3'-box RNA processing signal requires a novel phosphorylation mark on the pol II CTD. This new mark plays a critical role in the recruitment of the snRNA gene-specific RNA-processing complex, Integrator. These new findings provide the first example of a phosphorylation mark on the CTD heptapeptide that can be read in a gene-type-specific manner, reinforcing the notion of a CTD code. Here, we review the control of expression of snRNA genes from initiation to termination of transcription.