The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast

Abstract

Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.

Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex

“…In a recent paper, Wier and colleagues demonstrated that the interaction between the Rtf1 Plus3 domain and phospho-Spt5 is crucial for the recruitment of the PAF1C in S. cerevisiae (35). In S. pombe, however, Ctr9 is recruited independently of Rtf1 (30). Similarly, we found that human Paf1 was recruited to actively transcribed genes, apparently in an Rtf1-independent manner (Fig.…”

“…In vivo, Mueller and Jaehning (2) showed that many of the phenotypes associated with paf1⌬ or ctr9⌬ are not enhanced but, rather, are suppressed by simultaneous deletion of RTF1 in S. cerevisiae. Another recent paper showed that Rtf1 and the PAF1C exert opposing effects on Pol II elongation in S. pombe (30). Figure 7 suggested that the PAF1C is recruited to some of its target genes independently of Rtf1.…”

“…Purification of the PAF1C from human HeLa cells yielded a five-subunit complex lacking Rtf1 but containing Ski8 as an additional subunit (22,26,27). Similarly, coimmunoprecipitation studies in zebrafish, Drosophila, and Schizosaccharomyces pombe showed that PAF1C homologs in these species lack a stably associated Rtf1 subunit (28)(29)(30). At the functional level, however, Rtf1 and other PAF1C subunits have a number of similarities.…”

Genome-wide Screening of Regulators of Catalase Expression

“…This seems to indicate that the Paf1 complex exerts a positive role on transcription. Of the three deletion strains tested (lacking Prf1/ Rtf1, Paf1, or Leo1-coding genes), the loss of Prf1/Rtf1 affects tolerance to H 2 O 2 to a lower extent than a lack of the other two components, consistent with a recent report indicating that this protein may not belong to the core of the Paf1 complex in S. pombe (25).…”

Chromatin modification by the RNA Polymerase II elongation complex

“…[14][15][16][17] Interestingly, capping enzyme interactions with the Spt5 CTD are abolished by phosphorylation, whereas binding of the histone modification co-factor Rtf1 is enhanced. 13,21,24,25 As such, phosphorylation of the Spt5 CTD is beginning to emerge as an important part of the transition from early to late elongation.…”