RNA Polymerase II Carboxy-Terminal Domain Phosphorylation Is Required for Cotranscriptional Pre-mRNA Splicing and 3′-End Formation

Abstract

We investigated the role of RNA polymerase II (pol II) carboxy-terminal domain (CTD) phosphorylation in pre-mRNA processing coupled and uncoupled from transcription in Xenopus oocytes. Inhibition of CTD phosphorylation by the kinase inhibitors 5,6-dichloro-1␤-D-ribofuranosyl-benzimidazole and H8 blocked transcription-coupled splicing and poly(A) site cleavage. These experiments suggest that pol II CTD phosphorylation is required for efficient pre-mRNA splicing and 3-end formation in vivo. In contrast, processing of injected pre-mRNA was unaffected by either kinase inhibitors or ␣-amanitin-induced depletion of pol II. pol II therefore does not appear to participate directly in posttranscriptional processing, at least in frog oocytes. Together these experiments show that the influence of the phosphorylated CTD on pre-mRNA splicing and 3-end processing is mediated by transcriptional coupling.In eukaryotic cells, pol II synthesizes pre-mRNA that is processed in the nucleus to become mature mRNA and is then exported to the cytoplasm. Capping, splicing, and 3Ј-end processing are interdependent and often occur cotranscriptionally on the nascent transcript at the DNA template (2, 5, 32). Processing can also occur posttranscriptionally after release from the site of transcription (3, 37). The carboxy-terminal domain (CTD) of the largest subunit of pol II (Rpb1) provides an important link between transcription and processing by acting as a landing pad that binds directly to processing factors and localizes them to the site of transcription (4,7,13,14,23,31). In mammalian cells, pol II lacking the CTD produces transcripts that are not efficiently capped, spliced, or cleaved at poly(A) sites (24,25). Furthermore, in vitro the CTD can enhance capping, splicing, and poly(A) site cleavage uncoupled from transcription (15-17, 33, 40, 42, 43). These results suggest that the CTD of pol II that is not transcriptionally engaged can act as an allosteric activator of pre-mRNA processing reactions. Although the CTD is important for pre-mRNA processing, pol II transcription is by no means essential. RNA precursors can be processed in vitro and, in some cases, in vivo in the absence of transcription. Introns appearing early in the premRNA of Chironomus BR1 and BR3 genes are predominantly spliced at the site of transcription, whereas introns close to the 3Ј end are spliced after the transcript has been released (3, 37). It is also possible that cleavage and polyadenylation occurs posttranscriptionally, because cleavage frequently does not precede termination (29). It is not known if pol II that is not transcriptionally engaged can facilitate pre-mRNA processing in vivo after release from the site of transcription.During transcription, the CTD undergoes extensive phosphorylation and dephosphorylation on Ser2 and Ser5 residues of the heptad repeats (YSPTSPS). CTD hyperphosphorylation by CDK7 and CDK9 is associated with the transition from initiation to elongation (19,21). Protein kinase inhibitors, including 5,6-dichloro-1␤-D-ribofuranosyl...

Structural and functional characterization of an atypical activation domain in erythroid Krüppel-like factor (EKLF)

“…Recent studies have demonstrated that TFIIH is a rate-limiting factor during the elongation phase of β-globin gene expression (28,29), and this is consistent with the fact that patients with mutations in TFIIH suffer from β-thalassemia (30). Together, these studies indicate that TFIIH plays a crucial role in β-globin gene expression.…”

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes

“…Inhibitors of Cdk9 cause elongational arrest and can disrupt cleavage/polyadenylation 38,39 , but it is not clear how they affect recruitment of 3′ end processing factors. We investigated how DRB affects pol II, CstF and CPSF localization.…”

“…CTD S2 phosphorylation is usually regarded as a signature of elongating pol II within a gene 5, 7 however our results suggest a role for this modification well downstream of the gene where 3′ processing factors are recruited. How DRB inhibits cleavage/polyadenylation 38,39 is not completely resolved, but it correlates with both reduced recruitment of CstF and CPSF in 3′ flanking regions and inhibition of 3′ pausing (Figs. 6B, C, Supplementary Table 2).…”

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

“…First, transcription may target RNA to splicing, ensuring that transcribed introns will be recognized, committed to the splicing pathway, and removed, resulting in an improved overall efficiency of splicing. Such an effect has been observed in vivo, where splicing of a synthetic RNA injected into X. laevis oocytes was less efficient than cotranscriptional processing (Bird et al 2004). Second, splicing kinetics may be improved, if the transcription elongation complex delivers some or all components of the spliceosome to splicing signals; this could reduce the time needed for splicing complex formation and speed up all the steps of the process that precede the catalytic steps.…”