Spt5 phosphorylation and the Rtf1 Plus3 domain promote Rtf1 function through distinct mechanisms

Chen, J. J.; Mbogning, J.; Hancock, M.; Majdpour, D.; Madhok, M.; Nassour, H.; Dallagnol, J. C.; Page, V.; Chatenet, D.; Tanny, J. C.

doi:10.1101/2020.04.15.044131

bioRxiv · 10.1101/2020.04.15.044131

Spt5 phosphorylation and the Rtf1 Plus3 domain promote Rtf1 function through distinct mechanisms

Chen, J. J.·Mbogning, J.·Hancock, M.·Majdpour, D.·Madhok, M.·Nassour, H.·Dallagnol, J. C.·Page, V.·Chatenet, D.·Tanny, J. C.

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Abstract

Rtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes co-transcriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), 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 with 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.

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Chen, J. J., Mbogning, J., Hancock, M., Majdpour, D., Madhok, M., Nassour, H., Dallagnol, J. C., Page, V., Chatenet, D., Tanny, J. C.. 2020-04-17. Spt5 phosphorylation and the Rtf1 Plus3 domain promote Rtf1 function through distinct mechanisms. https://doi.org/10.1101/2020.04.15.044131

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