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

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...

Protein factors in pre-mRNA 3′-end processing

“…4A), which prefers the phosphorylated form of the CTD [157,172,173]. The crystal structure of Pcf11p in complex with a phosphorylated CTD peptide has been reported (Fig.…”

“…Additionally, yeast CTD interacts with Cft2p/Ydh1p and Pcf11p [118,172,173,213] and the mammalian CTD binds CstF-50 [85]. Protein binding generally increases upon phosphorylation of the CTD, and 3′-end processing is stimulated in vitro by phosphorylated CTD [215].…”

Transcription termination by nuclear RNA polymerases

“…Ser2 phosphorylated CTD helps to recruit and/or stabilize polyadenylation factors, thereby facilitating coupling of transcription and mRNA 39-end formation. For instance, in yeast, Ser2 phosphorylation of the CTD potentiates interaction with the essential polyadenylation factor Pcf11 (de Vries et al 2000;Barilla et al 2001;Licatalosi et al 2002;Ahn et al 2004). The H3-K36 methyltransferase Set2 binds elongating RNAPII, recognizing the doubly phosphorylated CTD (Ser5/Ser2) (Hampsey and Reinberg 2003;Kizer et al 2005).…”

“…For example, the C-terminal domain (CTD) of the RNAPII largest subunit also plays an important role in 39-end processing, likely by mediating interactions with 39-end processing factors (McCracken et al 1997;Hirose and Manley 1998;Barilla et al 2001;Fong and Bentley 2001). The Paf1 complex (Paf1C), which was first identified in yeast as an elongation factor and plays a role in transcription-associated chromatin modification (Krogan et al 2002;Squazzo et al 2002;Simic et al 2003;Mueller et al 2004;Rozenblatt-Rosen et al 2005), has been shown to be involved in mRNA 39-end formation (Penheiter et al 2005;Rosonina and Manley 2005) by interacting with CPSF, CstF, and symplekin (Nordick et al 2008;Rozenblatt-Rosen et al 2009).…”

RNA polymerase II carboxy-terminal domain with multiple connections

“…The 3'-end processing factors can bind to the CTD affinity column (McCracken et al, 1997). Furthermore, several factors, including Pcf11, Pta1, and Rna14 show an apparent preference for binding to the phosphorylated CTD (Rodriguez et al, 2000;Barilla et al, 2001;Licatalosi et al, 2002;Meinhart and Cramer, 2004). In particular, the yeast 3'-end processing factors appear to be recruited in time through the phosphorylation of S2 of the CTD when pol II approaches the 3'-end of a gene.…”