Role of the C-Terminal Domain of RNA Polymerase II in U2 snRNA Transcription and 3′ Processing

Abstract

U small nuclear RNAs (snRNAs) and mRNAs are both transcribed by RNA polymerase II (Pol II), but the snRNAs have unusual TATA-less promoters and are neither spliced nor polyadenylated; instead, 3 processing is directed by a highly conserved 3 end formation signal that requires initiation from an snRNA promoter. Here we show that the C-terminal domain (CTD) of Pol II is required for efficient U2 snRNA transcription, as it is for mRNA transcription. However, CTD kinase inhibitors, such as 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), that block mRNA elongation do not affect U2 transcription, although 3 processing of the U2 primary transcript is impaired. We show further that U2 transcription is preferentially inhibited by low doses of UV irradiation or actinomycin D, which induce CTD kinase activity, and that UV inhibition can be rescued by treatment with DRB or H7. We propose that Pol II complexes transcribing snRNAs and mRNAs have distinct CTD phosphorylation patterns. mRNA promoters recruit factors including kinases that hyperphosphorylate the CTD, and the CTD in turn recruits proteins needed for mRNA splicing and polyadenylation. We predict that snRNA promoters recruit factors including a CTD kinase(s) whose snRNA-specific phosphorylation pattern recruits factors required for promoter-coupled 3 end formation.RNAs that encode proteins are transcribed by RNA polymerase II (Pol II) in almost all eukaryotes. In contrast, untranslated RNAs are transcribed by all three RNA polymerases: 5.8, 18, and 28S rRNA by Pol I; 5S rRNA, tRNA, and U6 small nuclear RNA (snRNA) by Pol III (56); and the other U snRNAs, which function in mRNA splicing and various RNA processing events, by Pol II (27). Kinetoplastid protozoa, a class of early diverging eukaryotes, are exceptions to these rules. Kinetoplastid snRNAs are transcribed not by Pol II but by Pol III (65), and certain mRNAs, including the immunologically important variant surface glycoprotein message, are hybrids of a U snRNA-like spliced leader transcribed by Pol II and a protein-coding mRNA body transcribed by Pol I (19).Although U snRNAs and mRNAs are both transcribed by Pol II in mammals, the genes are very different. U snRNA promoters have no TATA box and rely instead upon a dedicated U snRNA-specific promoter consisting of a highly conserved proximal sequence element (PSE) and an enhancer-like distal sequence element spaced one nucleosome apart (27). In addition, U snRNA genes are short (typically only a few hundred base pairs) and lack introns, whereas genes encoding mRNAs can span megabases and usually contain many introns. Also, U snRNA genes are typically present in multiple copies in higher eukaryotes-the human U1 and U2 genes are tandemly repeated (6, 40, 66, 68)-whereas most protein-coding genes are present in only one or a few copies per haploid genome.U snRNA processing also differs from mRNA processing. U snRNAs are neither spliced nor polyadenylated; instead, formation of the first U snRNA interme...

Formation of the 3′ end of histone mRNA: Getting closer to the end

“…Pre-snRNAs are processed by a 12-subunit complex called Integrator that contains a putative endonuclease RC-68/Int11 and directly associates with the CTD (Baillat et al, 2005). Inhibition of Cdk9 activity does not affect generation of pre-snRNAs although it abolishes their 3' end processing (Jacobs et al, 2004;Medlin et al, 2005;Medlin et al, 2003), demonstrating that the CTD phosphorylated on serine 2 is selectively required for 3' end processing of pre-snRNAs but is dispensable for transcription elongation. In agreement with these results, addition of the phosphorylated CTD stimulates in vitro 3' end processing of snRNA precursors .…”

Transcription termination by nuclear RNA polymerases

“…CTD truncation and CTD kinase inhibitors affect 39-box recognition of U2 but not transcription termination (Medlin et al 2003;Jacobs et al 2004). Medlin et al (2005) showed that P-TEFb and phosphorylation of both Ser2 and Ser5 are required for cotranscriptional recognition of the 39-box.…”

Cooperation between Small Nuclear RNA-activating Protein Complex (SNAPC) and TATA-box-binding Protein Antagonizes Protein Kinase CK2 Inhibition of DNA Binding by SNAPC

“…As a negative control, RNAi was also performed using lacZ-specific RNA. Because it was demonstrated that phosphorylation of the carboxyl-terminal domain of RNA polymerase II contributes to 3Ј processing of human U2 snRNA (48,49), it was possible that CK2 could also play a role in U1 3Ј processing. Therefore, in this experiment different primer combinations were used to detect either the primary U1 snRNA transcript that is normally processed rapidly or the total U1 snRNA steady state population.…”