Direct Interactions between the Paf1 Complex and a Cleavage and Polyadenylation Factor Are Revealed by Dissociation of Paf1 from RNA Polymerase II

In Saccharomyces cerevisiae, the Paf1 complex (Paf1C), composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, is found associated with RNA polymerase II (Pol II) from the promoters to the poly(A) sites of actively transcribed genes (23, 31, 32). The homologous complex in humans is composed of human Paf1 (hPaf1), hCtr9, hCdc73, and hLeo1 (45, 56, 60). Although there is an hRtf1 homolog, it does not appear to be part of the hPaf1C. The yeast Paf1C is recruited to Pol II at an as-yet-uncharacterized early step in the transcription cycle, subsequent to initiation complex formation (16), which is dependent on the Bur1 kinase (27). The presence of Paf1C is required for the histone H2B monoubiquitylation activity of Rad6; the modified histone H2B subsequently serves as the necessary substrate for H3 lysine 4 (K4) methylation by Set1 and H3 K79 methylation by Dot1 (27, 33, 54, 55). Therefore, the loss of the Paf1C ultimately results in changes in histone modifications. Transcription by Pol II includes a cycle of phosphorylation and dephosphorylation of the C-terminal domain (CTD) of the largest Pol II subunit (reviewed in references 4, 7, 36, 39, 42, and 61). The initiating form of the enzyme is unphosphorylated; initiation and promoter escape are associated with, although not dependent on (21), phosphorylation of serine 5 (Ser5) of the YSPTSPS repeat of the CTD. During elongation, there is additional phosphorylation of serine 2 (Ser2) of the repeat. At, or just after, termination, the CTD is dephosphorylated, resetting Pol II for another transcription cycle. These modifications create unique interaction sites for a large and growing collection of transcriptional and posttranscriptional factors, including the mRNA-capping enzymes, histone modification enzymes, and the factors required for mRNA 3′-end formation and transcription termination (4, 6, 58). With these CTD modifications in mind, it is interesting to consider the Paf1C, which in both its yeast and human forms has been shown previously to associate with all of the various forms of Pol II (32, 45) and whose association with Pol II and chromatin in yeast is unaffected by the loss of Ser2 phosphorylation (Ser2-P) caused by the loss of the major CTD Ser2 kinase Ctk1 (1, 10, 37). In contrast, loss of the Paf1C results in a reduction of CTD Ser2-P on transcribed genes and a concomitant decrease in the association of a Ser2-P-dependent factor (Pcf11) (32) and, by extension, all factors sensitive to CTD Ser2-P. Of particular interest in this category are the polyadenylation factors, several of which have been shown previously to interact directly with the Ser2-P form of the CTD (reviewed in references 6, 8, and 43). Yeast and humans have similar complexes involved in mRNA 3′-end formation (reviewed in references 8, 12, 43, 48, and 59). Yeast CPF is a subcomplex of cleavage and polyadenylation factors, including Cft1 (Yhh1) and Ydh1 (Cft2), which is homologous to the human cleavage and polyadenylation factor subcomplex CPSF. CPSF is known to make direct contacts with RNA sequences at the poly(A) site. The yeast CF1A complex, which includes Pcf11, is similar to human CstF. Recently, evidence implicating human CPSF-73, the homolog of Ydh1, as the endonuclease responsible for cleavage has accumulated (15, 46), and Pcf11 was found previously to be able to directly trigger termination by purified Pol II (58). Cft1 and Pcf11 are among the factors reported previously to associate directly with the Ser2-P form of the Pol II CTD (3, 14, 26, 28), establishing physical links between both the CPF and CF1A complexes and Pol II. Therefore, posttranscriptional 3′-end formation is linked to transcription both by direct interactions with the Pol II CTD and by affinity for sequences in the nascent mRNA. The fact that the Paf1C is necessary for full levels of CTD Ser2-P on actively transcribed genes (32) provides an excellent explanation for why mutations in the Paf1C factors result in defects in posttranscriptional processing. The loss of Paf1 results in shortened poly(A) tails (32) and, for a subset of genes, changes in poly(A) site utilization and the formation of unstable transcripts (38). In addition, the Paf1C is critical for the proper formation of 3′ ends of nonpolyadenylated RNAs (49). However, there is evidence that the connection between the Paf1C and polyadenylation factors may be more than the indirect consequence of reduced CTD Ser2-P. For example, the loss of Rtf1 results in only partial reduction of CTD Ser2-P levels (32) but causes defects in the formation of 3′ ends of nonpolyadenylated RNAs (49). In addition, the loss of either Paf1 or Rtf1 is synthetically lethal in combination with the loss of Ctk1 (13), which implies that the Paf1C has functions in addition to its effects on Ser2-P. In this work, we demonstrate that the Paf1C interacts in a Pol II-independent fashion with the polyadenylation factor Cft1. We also found that the presence of the Paf1C on Pol II was critical for the association of Cft1 with the Ser5-P form of Pol II. We conclude that the Paf1C helps to recruit the polyadenylation factor Cft1 and that it forms an additional early point of contact for the assembly of an active polyadenylation complex on the elongating Pol II.