Repression of Meiotic Gene Expression by Regulated RNA Stability and by Antisense Transcription in Schizosaccharomyces pombe

Abstract

Meiosis produces haploid gametes from diploid germ cells. Many genes are used only for meiosis and cause harm if they are expressed in vegetative (non-meiotic) cells. My dissertation research focused on mechanisms that keep meiotic functions completely off in vegetative cells in a model organism, the fission yeast Schizosaccharomyces pombe. Early meiotic genes drive a specialized S-phase and meiotic recombination events. I found that at least 30 early meiotic genes in S. pombe are transcribed even in vegetative cells where they are kept off by degradation of their transcripts through a novel mechanism directed by Mmi1, an RNA binding protein. I determined that Mmi1 induces hyperadenylation of early meiotic genes, that the exonuclease Rrp6 degrades the hyperadenylated mRNAs, and that the polyA binding protein, Pab2, assists this hyperadenylation and is required for degradation. My work also shows that Mmi1 regulates mRNA splicing. These findings suggest that a polyadenylation quality control mechanism together with splicing regulation regulate early meiotic genes in S. pombe. Middle meiotic genes direct the specialized meiotic division events. Unlike the early meiotic genes, the middle meiotic genes are not transcribed in vegetative cells but are transcriptionally induced just before the first meiotic division. Using Affymetrix tiling arrays, I found that at least 50 middle meiotic genes are transcribed in the anti-sense direction in vegetative cells. Most of these anti-sense RNAs decline coincident with induction of the corresponding sense transcript during meiosis. To test the idea that anti-sense transcription inhibits expression of middle meiotic genes in vegetative cells, I engineered constructs to block three of the anti-sense RNAs. Using these constructs I found that disruption of the anti-sense RNAs allows sense gene transcription, and I determined that anti-sense mediated repression works together with the forkhead transcription factor, Fkh2 to keep middle meiotic genes fully off in vegetative growth. This thesis reports my experiments on mechanisms for repression of meiotic genes in vegetative cells including extensive characterization of RNA processing and decay pathways for repression of early meiotic genes, as well as experiments on anti-sense based repression of middle meiotic genes.