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

Abstract

The Paf1 complex (Paf1, Ctr9, Cdc73, Rtf1, and Leo1) is normally associated with RNA polymerase II (Pol II) throughout the transcription cycle. However, the loss of either Rtf1 or Cdc73 results in the detachment of the Paf1 complex from Pol II and the chromatin form of actively transcribed genes. Using functionally tagged forms of the Paf1 complex factors, we have determined that, except for the more loosely associated Rtf1, the remaining components stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin. The loss of Paf1, Ctr9, or to a lesser extent Cdc73 or Rtf1 results in reduced levels of serine 2 phosphorylation of the Pol II C-terminal domain and in increased read through of the MAK21 polyadenylation site. We found that the cleavage and polyadenylation factor Cft1 requires the Pol II-associated form of the Paf1 complex for full levels of interaction with the serine 5-phosphorylated form of Pol II. When the Paf1 complex is dissociated from Pol II, a direct interaction between Cft1 and the Paf1 complex can be detected. These results are consistent with the Paf1 complex providing a point of contact for recruitment of 3-end processing factors at an early point in the transcription cycle. The lack of this connection helps to explain the defects in 3-end formation observed in the absence of Paf1.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-yetuncharacterized 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 ...

RNA Polymerase II Carboxyl-terminal Domain Phosphorylation Regulates Protein Stability of the Set2 Methyltransferase and Histone H3 Di- and Trimethylation at Lysine 36

“…Prelich and co-workers demonstrated several years ago that deletion of several PAF complex members, cdc73⌬, ctr9⌬, or paf1⌬, resulted in a decrease in H3K36me3 (49). Similarly, Jaehning and co-workers showed that CTD Ser 2 phosphorylation is reduced in these same three PAF mutants (44). Our data show that Set2 protein levels are also decreased in cdc73⌬, ctr9⌬, and paf1⌬ strains (Fig.…”

“…Set2 protein levels were noticeably decreased in mutants of the PAF complex, which is known to play an important role in transcription elongation and affect CTD Ser 2 phosphorylation (Fig. 3B) (44). Significantly, Set2 protein levels were only decreased in PAF mutants that also exhibited changes in CTD Ser 2 phosphorylation, cdc73⌬, paf1⌬, and ctr9⌬.…”

Small region of Rtf1 protein can substitute for complete Paf1 complex in facilitating global histone H2B ubiquitylation in yeast

“…Previous studies showed that Paf1 and Ctr9 are important for Paf1C stability and integrity (26,28). To eliminate the residual Paf1C that forms in the absence of Rtf1 (29) and test the effect on HMD activity, we transformed the NLS-Myc-HMD expression plasmid into rtf1Δ paf1Δ and rtf1Δ ctr9Δ double mutants. Remarkably, even in the absence of Paf1 or Ctr9, the HMD behaved similarly to fulllength Rtf1 and was sufficient to direct H2B K123 ubiquitylation, H3 K79 dimethylation/trimethylation, and H3 K4 dimethylation, although at a reduced level compared with strains containing both Paf1 and Ctr9 (Fig.…”

“…Paf1C-which in budding yeast consists of the subunits Paf1, Ctr9, Cdc73, Rtf1, and Leo1-impacts RNA synthesis at multiple stages (21). Paf1C associates with RNA pol II from the 5′ end of a gene to the poly(A) site (22,23); interacts functionally and physically with the transcription elongation factors Spt4-Spt5/DSIF, Spt16-Pob3/FACT, and TFIIS (8,(24)(25)(26)(27); regulates the phosphorylation state of RNA pol II (28,29); and is required for proper 3′ end formation of certain transcripts (30)(31)(32). Important to this study, deletion of RTF1 from yeast cells causes dramatic reductions in H2B K123 ubiquitylation and H3 K4 and K79 methylation (33)(34)(35)(36), and this role in histone modification, like other Paf1C functions, is conserved in higher eukaryotes (37)(38)(39)(40).…”

Transcription termination by nuclear RNA polymerases

“…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). It is notable that one Paf1C subunit, Cdc73 or parafibromin, has been identified as a tumor suppressor protein, and the Paf1 protein is overexpressed in many cancers (Chaudhary et al 2007).…”