pac1+ FUNCTION IN THE CONTEXT OF MEIOSIS AND STRESS RESPONSE

Abstract

S. pombe is an excellent model system to study the biogenesis and function of non-coding RNAs (ncRNAs), RNA processing, RNA interference (RNAi) and heterochromatin right down to the molecular details because these systems are conserved with humans. Here, I have investigated the function of pac1+ that encodes an anti-meiotic RNase III nuclease necessary for structural RNA biogenesis.At a cellular level, I found that changes in Pac1 activity not only affected meiosis (differentiation) but also the tolerance to oxidative stress. To study the involvement of pac1+ in these cellular programs, I carried out the integration of total RNA, poly(A) RNA and small RNA sequencing data of strains overexpressing pac1+ alongside pac1 mutant strains (T301>N, I305>T, G328>C) in which growth is abrogated at high temperatures. This analysis revealed aberrant RNA processing profiles of known Pac1 targets and a new set of transcripts. These transcripts could potentially be direct targets of Pac1 enzymatic activity. Inaccurate processing of structural RNAs coincides with raised levels of polyadenylated transcripts. I propose that the polyadenylated RNAs could be targets of a poly(A)-dependent degradation mechanism carried out by the TRAMP complex, an RNA quality surveillance mechanism. Incorrect processing of at least three snRNAs occurred in the pac1 mutant strain, which might explain the high number of intron retention events observed. Failure to splice introns correctly affects the coding potential of protein-coding genes and can be cellular programs such as meiosis and stress response.The pac1 mutant here analysed also exhibited a general induction of protein-coding transcripts (protein-coding and non-coding). For example, pac1 mutant diploid strains carrying mutations in pac1+ exhibited an induction of meiotic transcripts that could explain the deregulation of the sexual differentiation program. Furthermore, it is yet not clear how pac1+ influences the stress response, however, the enhanced expression of pac1+ caused significant changes in nucleolar proteins that are related with ribosomal biogenesis. Thus, Pac1 protein may localise to the nucleolus, a protein-RNA centre of ribosomal biogenesis that responds to cellular programming, for example, stress response.Notably, the ncRNA network was profoundly affected in the pac1 mutant. Using correlation analysis, a subset of ncRNAs dependent on Pac1 activity was found associated with structural RNA biogenesis, stress response and meiosis. These data supports the increasing amount of evidence that ncRNAs are tightly regulated in response to external factors, and that they are critical to adapt to external challenges and differentiation. Further validation of the function of these ncRNAs and the role of Pac1 protein in the control of the ncRNA network are necessary.In general, this work contributed to the understanding the role of structural RNAs and ncRNAs in fission yeast, and how these pathways are integrated to respond to stress and differentiation; systems valid for the interrogation of pathogenesis such as cancer in higher eukaryotes.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Iain Hagan (Supervisor) & Crispin Miller (Supervisor)