Title: Elucidating the functions of the RNA polymerase II elongation factor Spt5 through a multidisciplinary approach
Abstract:
Transcription elongation by RNA polymerase II (RNAPII) is a highly dynamic and regulated step of the gene expression cycle. Transcription elongation factors form an elongation complex with RNAPII and help coordinate chromatin states with mRNA synthesis during elongation. Spt5 is an essential and highly conserved protein that promotes elongation by maintaining RNAPII processivity. Spt5 associates with RNAPII from early elongation until termination and has been implicated in the regulation of various co-transcriptional processes such as chromatin maintenance and mRNA processing. Previous work from our lab and others described a role for Spt5, specifically its C-terminal repeat domain (CTR), in the recruitment of Polymerase Associated Factor 1 complex (Paf1C) to form the active elongation complex. The Spt5-CTR is a conserved eukaryotic feature of Spt5 that gets dynamically phosphorylated during transcription elongation. However it is dispensable for viability of yeast cells and its functional significance in transcription is not well understood. I am employing a multidisciplinary approach to comprehensively characterize the functions of the Spt5-CTR. First, I performed chromatin immunoprecipitation (ChIP) assays followed by deep sequencing on strains acutely depleted of Spt5 or a panel of targeted spt5-CTR mutants. Rapid depletion of Spt5 caused a widespread severe impact on RNAPII elongation and the assembly of the elongation complex. The results from the mutants revealed an important role for the Spt5-CTR in this assembly. To uncover novel functions of the Spt5-CTR, I have performed a transposon-based genetic screen in yeast to identify its genetic interactors. Strikingly, I found that mutations in the Spt5-CTR ablating its phosphorylation can suppress the lethality of deleting essential members of the cleavage and polyadenylation factor (CPF). Simultaneously, I have performed site-specific in vivo photo crosslinking experiments to profile the physical interactome of the Spt5-CTR. From pilot mass spectrometry analyses, a known interaction between a Paf1C subunit and the Spt5-CTR has been detected, confirming the validity of this approach. Collectively, these studies reveal the functional importance of Spt5 and its phosphorylation in the assembly and dynamics of the RNAP II elongation complex and in the transition from elongation to termination.
Arndt Lab
Friday, February 9th, 2024
12:00PM
Langley A219B