Asymmetric Regulation of Gene Expression in S. Cerevisiae through RNA Localization

Intellectual Merit

Development of a multi-cellular organism requires that a mother cell divide into two daughter cells with different cell-fates. The ability of the two daughter cells to assume different developmental potentials is determined by the asymmetric regulation of gene expression. The asymmetric sorting of a cell-fate determinant between the daughter cells leads to differences in gene expression. RNA localization is one mechanism for asymmetrically sorting a cell-fate determinant between daughter cells. The yeast S. cerevisiae serves as a model system to investigate the asymmetric sorting of a cell-fate determinant through mRNA localization. ASH1 mRNA localizes to the distal tip of the bud resulting in the asymmetric regulation of gene expression by the transcriptional repressor Ash1p. The localization of ASH1 mRNA is achieved through the interplay of four cis-acting localization elements located in ASH1 mRNA and at least three (Myo4p, She2p and She3p) trans-acting localization factors. The various protein-RNA and protein-protein interactions between the cis- and trans-acting factors have led to a very basic model for ASH1 mRNA localization which does not completely account for all aspects of the ASH1 mRNA localization pathway. There is increasing evidence indicating that phosphorylation plays a role in the mechanism of RNA localization. She3p is phosphorylated in vivo, and phosphorylation of this protein negatively regulates a novel activity required for ASH1 mRNA localization. Consequently, one aim of this project is to identify and characterize this novel She3p activity and determine how phosphorylation of She3p negatively regulates this activity. The functions of Myo4p and She3p in the yeast cell are not limited to RNA localization. Myo4p and She3p participate in the delivery of cortical endoplasmic reticulum (ER) to the bud. Furthermore, a fraction of She2p co-fractionates with the ER. Given the correlation between the ER and ASH1 mRNA localization components, a second aim of this project is to determine if there is a mechanistic connection between ASH1 mRNA localization and the association of She2p with the ER. The biochemical, genetic and cell biological experiments contained within this project will further uncover the mechanism for ASH1 mRNA localization, and the insight obtained from S. cerevisiae will likely have relevance for the sorting of cell-fate determinants required for development in higher eukaryotes.

Broader Impacts

Research in Dr. Long's laboratory is heavily dependent on research conducted by post-doctoral fellows and graduate students. Since arriving at the Medical College of Wisconsin, Dr. Long has trained two post-doctoral fellows. One of these fellows is currently an Assistant Professor at Loyola-Marymount University. Dr. Long has also trained two graduate students who have garnered post-doctoral positions in excellent laboratories at the University of North Carolina and the University of Miami Miller School of Medicine. Furthermore, in the Long laboratory a third graduate student is currently pursuing her Ph.D. and a third post-doctoral fellow is acquiring additional skills and expertise. Dr. Long's laboratory has an established history of including undergraduate students within his research program by hosting five undergraduates in the laboratory for 10 weeks of research training during the summer. Dr. Long anticipates a similar level of student participation in the studies proposed in this application and expects that his undergraduate research training environment will increase through the recruitment of undergraduates from small liberal arts colleges in and surrounding Milwaukee. Dr. Long's laboratory also has a strong record of publishing in high visibility journals (Eukaryotic Cell, Journal of Biochemistry, Molecular Biology of the Cell and RNA) and participating in prominent meetings (American Society for Cell Biology and FASEB Summer Research Conferences). In addition to his classroom teaching responsibilities, Dr. Long's interest in scientific education is further reflected by his role as a Course Director in Classical and Molecular Genetics and previously in Techniques of Molecular and Cell Biology. Furthermore, Dr. Long serves on the Medical College of Wisconsin-Interdisciplinary Program (graduate student) Admissions Committee as well as the Medical College of Wisconsin Medical School Admissions Committee, and he also participates in guiding the direction of the Medical College of Wisconsin Graduate School by his active participation on the Graduate Studies Council.