In higher eukaryotic cells, RNA localization functions in establishing intracellular polarity and intercellular asymmetry. The long-term objective of the laboratory is establishing the mechanism controlling RNA localization. The yeast S. cerevisiae serves as a model system for studying the asymmetric segregation of mRNA. ASH1 mRNA is localized to the distal end of daughter cells during anaphase of the cell cycle, resulting in the exclusive deposition of Ash1p in daughter nuclei. Ash1p is a daughter cell specific repressor of HO transcription. Localization of ASH1 mRNA requires SHE 1-5 and components of the actin cytoskeleton. In addition, at least three cis-acting localization elements (E1, E2 and E3) have been identified within the ASH1 mRNA. Each of these cis-acting elements is sufficient to localize a reporter mRNA to daughter cells. None of the She proteins share amino acid homology to known RNA binding proteins. From preliminary three-hybrid analysis, two yeast proteins were identified as RNA binding proteins to E3. One of the proteins is She3p, and the other protein was designated She6p. Both of these proteins function in ASH1 mRNA localization.
In aim one, the RNA binding activity of these two proteins will be determined.
In aim two, the molecular interactions for these RNA binding proteins will be identified and characterized.
Specific aim three will determine if the three cis-acting elements perform redundant or mechanistically independent functions. This will be achieved by comparing and contracting the molecular composition of each element. The slow growth phenotype associated with she6 strains suggests that She6p functions in other cellular processes.
In specific aim four, a genetic approach will be used for the identification of cellular processes requiring She6p. The ASH1 mRNA is the only known mRNA localized to daughter cells.
In specific aim five, the extent of RNA localization to daughter cells will be determined. The mechanism governing mRNA transport and localization remain elusive. However, many of the mechanisms controlling other aspects of gene expression are highly conserved from yeast to man. Consequently, we expect that the mechanism regulating mRNA localization will also be conserved from yeast to higher eukaryotes. With the power of yeast genetics, we are poised for significant progress into the mechanism controlling RNA localization.
