Roy M. Long, PhD
Microbiology and Molecular Genetics
Medical College of Wisconsin
Assistant Dean for Graduate Recruitment
Research Focus: RNA Localization in Yeast
PhD: Pennsylvania State University (1994) Biological Chemistry
Using genetic, biochemical and cell biological approaches in the yeast S. cerevisiae, my laboratory is interested in studying mechanistic details of RNA localization, a process which post-transcriptionally regulates gene expression. In yeast ASH1 mRNA is localized to the distal end of the daughter cell during anaphase of the cell cycle, resulting in the exclusive deposition of Ash1p in daughter nuclei. Ash1p is a transcriptional repressor, and asymmetric sorting of Ash1p results in asymmetric regulation of transcription.
ASH1 mRNA contains four cis-acting localization elements sufficient to localize a heterologous reporter mRNA to daughter cells. One of these elements resides in the 3’-untranslated region (3’-UTR) while the remaining three elements reside in the coding region of the ASH1 mRNA. Besides these cis-acting elements, five trans-acting factors (SHE1-5) and the actin cytoskeleton are required for localization of ASH1 mRNA. Our laboratory has focused on deciphering mechanistic details related to She1p, She2p and She3p. We demonstrated that the type V myosin, She1p/Myo4p, directly transports ASH1 mRNA containing particles to daughter cells. We also reported that She2p is an RNA-binding protein that directly interacts with each of the ASH1 cis-acting localization elements. Since She3p has the ability to interact with both Myo4p and She2p, we hypothesized that She3p functions to interface Myo4p with the She2p-ASH1 mRNA complex. We demonstrated that a Myo4p-She3p-She2p complex does exist in vivo, and this heterotrimeric complex functions to transport ASH1 mRNA to the bud tip. The function of She3p in ASH1 mRNA localization is not limited to interacting with Myo4p and She2p. We identified mutants of She3p defective for ASH1 mRNA localization, yet these She3p mutants retain the ability to associate with Myo4p and She2p. Our analysis of these She3p mutants suggest that each is defective for associating with ASH1 mRNA, implying that She3p could directly contact the ASH1 cis-acting localization elements. In addition, our experimental evidence suggests that a molecular reorganization of the heterotrimeric complex is required for ASH1 mRNA to be anchored at the bud tip.
Model for ASH1 mRNA localization. She2p binds ASH1 mRNA co-transcriptionally. In the cytoplasm She3p binds ASH1 mRNA which recruits Myo4p, resulting in formation of the heterotrimeric complex. The heterotrimeric complex is responsible for transporting the cargo to daughter cells on the polarized actin cytoskeleton. Following transport to the bud tip, molecular reorganization of the transport complex results in the release of She2p which allows anchoring factors to associate with ASH1 mRNA, She3p and Myo4p. Once anchored ASH1 mRNA is competent for translation in the daughter cell, and newly synthesized Ash1p is imported into the daughter cell nucleus.
It has been shown that the asymmetric segregation of specific mRNA molecules is imperative for normal development of higher eukaryotic organisms. The mechanisms controlling mRNA transport and localization remain elusive. However, many of the mechanisms controlling other aspects of gene expression such as transcription, translation and splicing, are highly conserved from yeast to man. Consequently, we expect that the mechanisms regulating mRNA localization will also be conserved from yeast to higher eukaryotes. With the power of yeast genetics, we are poised to make significant progress in dissecting the mechanisms controlling mRNA localization.