Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomycescerevisiae

Abstract

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factors. We show here that the cleavage͞ polyadenylation factor IA of Saccharomyces cerevisiae directly contacts CTD. First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphorylated CTD. This interaction is confirmed for Rna15p by yeast two-hybrid analysis. Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins. These findings establish a direct interaction of cleavage͞polyadenylation factor IA with the CTD. Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p. Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript. In Saccharomyces cerevisiae the largest subunit of RNA polymerase II (pol II) contains a carboxyl-terminal domain (CTD) consisting of 26 tandem repeats of a heptapeptide module with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (1, 2). Two serines per heptad repeat may undergo reversible phosphorylation during each transcription cycle (3). The phosphorylation status of the CTD is correlated with different stages of the transcription cycle. Thus hypophosphorylated polymerase (IIA) is competent for initiation, whereas hyperphosphorylated polymerase (II0) is associated with transcription elongation. Increasing evidence has been provided in the last few years showing that the CTD acts as a direct physical link between transcription and nascent RNA processing: in mammals, the cleavage-polyadenylation specificity factor (CPSF) and the cleavage stimulation factor (CstF), as well as splicing factors and 5Ј capping enzyme, all bind to the CTD (4-9). Furthermore, CPSF and CstF copurify with pol II (4). The concept of a factor recruiting͞docking platform has emerged as one likely function of this peculiar polypeptide. Evidence for a more direct role of the CTD in polyadenylation has also been indicated (10).In S. cerevisiae, the proteins so far known to bind to the CTD are the Mediator complex (11, 12), the Elongator complex (13), proteins involved in 5Ј end capping of nascent RNA (8,14), and the Ess1 peptidylprolyl isomerase (15, 16). To date the interaction of cleavage͞polyadenylation factors with the CTD remains largely uncharacterized. In yeast 3Ј-end processing of pre-mRNA requires cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PFI), poly(A) polymerase, and the poly(A)-binding protein 1 (17-22). CF IA, which is involved in both cleavage and po...

Structure of a nucleotide-bound Clp1-Pcf11 polyadenylation factor

“…Pcf11 interacts directly with all the other CFIA components (14) and links the cleavage-polyadenylation machinery to the transcriptional elongation complex by coupling CFIA to the phosphorylated CtD of RNA polymerase (30–32). Furthermore, Pcf11 also binds to several components of other 3′ end processing complexes (27) so it seems likely that part of Pcf11 function is to coordinate cross talk between CFIA, RNA polymerase and the other polyadenylation factors.…”

RNA Polymerase II Elongation Control

“…Multiple lines of evidence indicate that the Pol II CTD, especially the Ser2-phosphorylated form, promotes transcription-coupled 3’ end formation through recruiting key components of the processing machinery to their target sites. For instance, the Pcf11 subunit of the yeast cleavage/polyadenylation factor IA (CFIA) complex specifically recognizes the phospho-Ser2 CTD (164, 165). Similarly, the binding of human cleavage stimulation factor CstF-64 to Pol II depends on Ser2 phosphorylation in the CTD (166).…”

“…see (164, 165), the bindings are likely enhanced in vivo by transcription elongation factors that track along with Pol II, resulting in more efficient recognition and cleavage at poly(A) sites (Figure 6). For example, the multifunctional PAFc was found to be required for transcription-coupled polyadenylation stimulated by the prototypical transcriptional activator GAL4-VP16 (167).…”

Delineating the Structural Blueprint of the Pre-mRNA 3′-End Processing Machinery

“…4B). The CID interacts with both unphosphorylated and phosphorylated RNAP II CTDs but has higher affinity for the latter (133,136,137). Surprisingly, the CID-CTD interaction is not necessary for 3= processing, but it is required for proper transcription termination (133,136).…”

“…The CTD is necessary for efficient polyadenylation both in vivo (33) and in vitro (34), but exactly how it promotes mRNA 3=-end formation is still not well understood. A platform role has been proposed since a number of 3= processing factors have been observed binding to the CTD, such as Ydh1 (50), Yhh1 (70), CstF-77/Rna14 (33, 133), CstF-50 (33), Pcf11 (133,134,136,137), and perhaps Rna15 (133) and Pta1 (164).…”