Sciences and Mathematics, College of
Biology, Department of
BURS Faculty Advisor
Rebecca Adams, PhD
The export of mRNA from the nucleus to the cytoplasm is a regulatory point that is essential to the pathway of gene expression in eukaryotic cells. The export of mRNA transcripts is mediated through selective doorways called the nuclear pore complexes (NPC). Additionally, there are proteins associated with the nuclear pore complex that assist in facilitating the export. This includes association with the export receptor, Mex67, which binds to the transcript and ferries it through NPCs. During cellular stress, such as heat shock, the export of housekeeping mRNA transcripts is halted, forcing these transcripts to remain inside the nucleus and prohibiting their translation. In these conditions, only specific mRNA transcripts, including mRNA encoding the molecular chaperone SSA4, are permitted to export, allowing recovery from stress. The molecular mechanism of this selective export is not understood. In this study, I have hypothesized that an unknown adaptor recruits Mex67 to permit selective SSA4 export during stress. To test this hypothesis, my goal is to identify this protein by developing a multi-copy suppression screen (MCS). To enable a phenotypic outcome for this screen, I have successfully generated a colorimetric SSA4 reporter plasmid that expresses the ADE3 ORF under the regulation of the SSA4 promoter with SSA4 5’ and 3’ UTR sequences. It was anticipated that this plasmid would allow cells to turn red only when SSA4 is induced and exported following stress. However, we observed leaky expression of the reporter, as cells were equally red with and without heat shock. Our current approach is to generate a reporter by genomic integration of the ADE3 ORF into the endogenous SSA4 genetic locus. Following this series of steps, we will analyze the color of cells following stress. Once successful, this reporter will be generated in a mutant strain that slows SSA4 export under stress so that an MCS can be conducted to identify proteins that restore selective SSA4 export. By examining the identified proteins, we can determine how they modulate the export of mRNA transcripts to be transcribed and translated into products that mediate cellular function.
Hatem, Zaid, "Generating a Colorimetric SSA4 Transcript Export Reporter For Multicopy Suppression Screen in S. cerevisiae" (2022). Belmont University Research Symposium (BURS). 98.