Spt5 Phosphorylation and the Rtf1 Plus3 Domain Promote Rtf1 Function through Distinct Mechanisms

ABSTRACT

Rtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes cotranscriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires the phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), the catalytic component of positive transcription elongation factor b (P-TEFb). Rtf1 recognizes phosphorylated Spt5 (pSpt5) through its Plus3 domain. Since Spt5 is a unique target of Cdk9 and Rtf1 is the only known pSpt5-binding factor, the Plus3/pSpt5 interaction is thought to be a key Cdk9-dependent event regulating RNAPII elongation. Here, we dissect Rtf1 regulation by pSpt5 in the fission yeast Schizosaccharomyces pombe. We demonstrate that the Plus3 domain of Rtf1 (Prf1 in S. pombe) and pSpt5 are functionally distinct and that they act in parallel to promote Prf1 function. This alternate Plus3 domain function involves an interface that overlaps the pSpt5-binding site and that can interact with single-stranded nucleic acid or with the polymerase-associated factor (PAF) complex in vitro. We further show that the C-terminal region of Prf1, which also interacts with PAF, has a similar parallel function with pSpt5. Our results elucidate unexpected complexity underlying Cdk9-dependent pathways that regulate transcription elongation.

KEYWORDS:

• Cdk9
• Rtf1
• Spt5
• transcription elongation

View publisher note:

Article of Significant Interest in This Issue

ACKNOWLEDGMENTS

We thank K. Gull for kindly providing TAT1 monoclonal antibody against alpha-tubulin, R. Fisher, B. Schwer, and S. Shuman for S. pombe strains, F. Robert for S. cerevisiae expressing RPB1-TAP, and members of the Tanny lab for helpful discussions.

This work was supported by the Canadian Institutes for Health Research (MOP-130362 to J.C.T. and MOP-142184 to D.C.) and the Natural Sciences and Engineering Research Council of Canada (RGPIN 03661-15 to J.C.T. and RGPIN-2015-04848 to D.C.). J.C.T. and J.J.C. acknowledge support from the Fonds de Recherche santé Quebec (chercheur boursier 33115; doctorat boursier 259846). The McGill SPR-MS Facility is supported by the Canada Foundation for Innovation.