Functional Interaction of Yeast Pre-mRNA 3′ End Processing Factors with RNA Polymerase II

Abstract

The RNA polymerase II CTD is essential for 3' end cleavage of metazoan pre-mRNAs and binds 3' end processing factors in vitro. We show genetic and biochemical interactions between the CTD and the Pcf11 subunit of the yeast cleavage/polyadenylation factor, CFIA. In vitro binding to Pcf11 required phosphorylation of the CTD on Ser2 in the YSPTSPS heptad repeats. Deletion of the yeast CTD reduced the efficiency of cleavage at poly(A) sites, and the length of poly(A) tails suggesting that it helps couple 3' end formation with transcription. Consistent with this model, the 3' end processing factors CFIA, CFIB, and PFI were recruited to genes progressively, starting at the 5' end, in a process that required ongoing transcription.

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

“…A common conclusion drawn from these observations is that the CTD is phosphorylated on serine 5 at or near the promoter, while CTD repeats on elongating Pol II carry their phosphates on serine 2 but not serine 5. Notwithstanding the fact that the specificities of these antibodies may not in fact be as unambiguous as widely supposed (8,16,32) and that their epitopes might be masked by (P)CTD-bound factors, serine 5 phosphorylation is also detected in the coding regions of genes. Along the yeast PMA1 gene, for example, the H14 epitope is detected at levels 30-40% of those found at the promoter (4), minimally implying that elongating Pol II carries phosphates on both serines 2 and 5 of the CTD.…”

“…These different CTD phosphorylation patterns serve as a guide for the timely recruitment to transcribing Pol II of factors that play a role in transcript biogenesis. For example, Ser5 phosphorylation is important for recruiting capping enzyme early in the transcription cycle (6,7), whereas Ser2 phosphorylation plays a role in recruiting Pcf11, a factor involved in 3′-end formation (8). Modulating the extent and pattern of CTD phosphorylation can thus be an effective way of regulating the CTD's affinity for numerous protein factors, as it can potentially generate a profuse array of different phosphoepitopes (9).…”

Protein factors in pre-mRNA 3′-end processing

“…Purified phosphorylated and unphosphorylated CTD activated a reconstituted cleavage reaction in vitro [212]. While the CTD is necessary for cleavage in mammals, it does not appear to be necessary for cleavage in yeast [213]. Deletion of the CTD in yeast produces unstable mRNAs that are degraded, but the degradation can be rescued by blocking the 5′-to-3′ exonuclease XRN1, indicating a problem with the 5′-end cap as opposed to 3′-end polyadenylation [214].…”

“…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].…”

Cross-Validation of the Structure of a Transiently Formed and Low Populated FF Domain Folding Intermediate Determined by Relaxation Dispersion NMR and CS-Rosetta

“…It is the pattern of phosphorylation during elongation that determines which set of factors can bind. For example, factors involved in pre-mRNA 5" capping are recruited at the beginning of elongation, when mainly S 5 is phosphorylated [67], and 3" cleavage and polyadenylation factors are recruited near the end of the gene, when only S 2 is phosphorylated [68]. Many other factors are recruited at some point during transcription, when both serines are phosphorylated, including mRNA splicing factors [69,70] and histone acetyl transferases [71].…”