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.
Highlights
Regulation of RNA polymerase II (RNAPII) transcription elongation affects the expression of many eukaryotic genes [1]
positive transcription elongation factor b (P-TEFb) was identified on the basis of its biochemical activity in overcoming promoter-proximal RNAPII pausing imposed by DRB-sensitivity inducing factor (DSIF; a complex of Spt4 and Spt5) and a negative elongation factor (NELF) [2,3,4,5]
Using fission yeast Schizosaccharomyces pombe as a model system, we have identified two Cyclin-dependent kinase 9 (Cdk9)-dependent pathways that modulate RNAPII elongation
Summary
Regulation of RNA polymerase II (RNAPII) transcription elongation affects the expression of many eukaryotic genes [1]. A central regulator of metazoan elongation is positive transcription elongation factor b (P-TEFb), a complex comprising cyclindependent kinase 9 (CDK9) and a cyclin partner. P-TEFb was identified on the basis of its biochemical activity in overcoming promoter-proximal RNAPII pausing imposed by DRB-sensitivity inducing factor (DSIF; a complex of Spt and Spt5) and a negative elongation factor (NELF) [2,3,4,5]. Cdk phosphorylates multiple proteins involved in elongation, including Spt and the Rpb subunit of RNAPII [13]. In addition to regulating elongation, these phosphorylations are implicated in the recruitment of chromatin-modifying enzymes and mRNA 39-end processing factors [11,14,15,16]. Cdk can phosphorylate and activate the histone H2B ubiquitin-conjugating enzyme RAD6, indicating multiple links between Cdk and chromatin modification [17,18]
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