Ssu72 and Rtr1 Serine 5 Phosphates and Their Role in NNS and CPF Transcription Termination

Abstract

Polyadenylation dependent transcription termination is dependent on the Cleavage and Polyadenylation Factor complex (CPF) which is essential for the termination and processing of mature RNA. Polyadenylation (PolyA) independent transcription termination is carried out by the NNS (Nrd1-Nab3-Sen1) termination pathway, which helps regulate termination and processing of non-coding RNA (ncRNA). The disruption of these pathways can impact expression of nearby genes, both protein coding and noncoding. Recruitment of termination pathway components is achieved through a domain unique to the largest subunit of RNA Polymerase II (RNAPII) referred to as the Cterminal domain (CTD), which contains a repeating heptad sequence, Y1S2P3T4S5P6S7, and acts as a docking site for transcription regulatory proteins. Ssu72 is a serine 5 phosphatase and an essential member of the CPF complex. Rtr1 is also a serine 5 phosphatase, but its mechanism of action is less well characterized. Both Rtr1 and Ssu72 regulate transcription machinery recruitment through control of the phosphorylation status of the CTD. My studies have focused on Rtr1 and Ssu72 mutants in yeast which show evidence of transcription termination related phenotypes. Chromatin immunoprecipitation of RNAPII followed by exonuclease treatment (ChIP-exo) studies provide evidence of RNAPII transcription continuing through termination sites at ncRNA genes as a result of a hyperactive Ssu72-L84F mutant, while an RTR1 knockout results in increased premature RNAPII transcription termination. Northern blots and RNA sequencing confirm premature transcription termination and decreased total RNA expression in the RTR1 knockout and increased length of ncRNA transcripts as well as total RNA expression in the Ssu72-L84F mutant. Mass spectrometry analysis has identified changes in the protein-protein interactions (PPI) within the CPF complex in the Ssu72-L84F mutant and decreased PPIs between different transcription machinery in RTR1 knockout cells. My results show that the CTD phosphatases Rtr1 and Ssu72 play unique roles in the regulation of RNAPII termination in eukaryotes.