Regulation of mid-meiotic RNA processing by forkhead factors in fission yeast

Case Western Reserve University, Cleveland OH

Investigators

Abstract

INTELLECTUAL MERIT: Cells have evolved elaborate mechanisms to ensure that genes encoding proteins or RNAs that are required only in specific cell types or at certain times in development are tightly regulated. This project will utilize the fission yeast Schizosaccharomyces pombe as a simple model system to gain broadly applicable insights into the molecular events responsible for maintaining strict temporal control over gene expression. Meiosis, a differentiation process that produces haploid gametes from diploid precursor cells through division without DNA synthesis, and mitosis, through which cells proliferate through division after DNA synthesis, are conserved pathways throughout the eukaryotic kingdom. In fission yeast, the switch from cellular proliferation to differentiation in response to adverse environmental conditions is mediated by sequential waves of altered transcript accumulation that correlate with early events (DNA replication and recombination), middle events (cell division) and late events (spore formation). The overarching goal of this project is to dissect the molecular mechanisms through which members of the forkhead family of transcription factors function antagonistically to prevent inappropriate expression of meiotic genes in mitotically growing cells and collaboratively to promote productive RNA synthesis and processing during meiosis. Although some forkhead family members act as conventional promoter-restricted transcription factors that regulate initiation, recent work in the principal investigator's laboratory has revealed association of these factors along the entire gene body and even downstream from the coding region in meiotic cells. Consistent with this unusual distribution, complementary data indicate that forkhead factors regulate the addition of a polyadenosine tail to newly synthesized mRNAs, which is mechanistically linked to transcription termination. The first two specific aims of this project will test the working hypotheses that the decision between polyadenylation and read-through transcription is enforced by differential recruitment of RNA processing factors at the promoter and/or competition between elongation and termination as the transcription complex traverses the polyadenylation signals. The third aim will utilize deep sequencing of RNA from forkhead-regulated meiotic genes, followed by bioinformatic analysis to reveal shared sequence motifs and other features that may be required for their coordinate regulation. The discovery that forkhead factors function at the interface between transcription and RNA processing resonates with recent developments in the gene expression field as a whole, and provides a unique opportunity to investigate polyadenylation, a vital but understudied regulatory mechanism, in a biologically relevant context. BROADER IMPACTS: This project will build upon the principal investigator's strong track record of mentoring scientists from diverse backgrounds and at various stages of professional development. The postdoctoral fellow will play a pivotal role by co-supervising undergraduate and high school students in addition to receiving training in cutting-edge experimental strategies. The collegial atmosphere in the Center for RNA Molecular Biology at CWRU will help to ensure that broader impact goals are achieved, as will the use of fission yeast, which resembles multicellular eukaryotes at the molecular level, yet is relatively simple to culture and manipulate even by students just beginning bench research. As in the past, a particular effort will be made to include women and members of groups generally under-represented in scientific research in the project.