Sciences and Mathematics, College of
Biology, Department of
BURS Faculty Advisor
The synthesis of genome-encoded proteins via mRNA translation is integral to cell survival. In eukaryotes, such as S. cerevisiae, the mRNA that is produced in the nucleus must be exported to the cytoplasm for translation to occur, and this process is highly regulated. Specifically, the export of mRNA occurs via travel through nuclear pore complexes (NPCs), which are selective doorways embedded in the nuclear envelope. During cellular stress, such as heat shock, the cell needs to regulate gene expression to permit survival, and mRNA export is one step at which this occurs. At these high temperatures, a cell’s proteins unfold, rendering them non-functional. However, a group of proteins that are induced in response to heat shock called chaperones, catalyze the refolding of proteins, restoring their function. For the selective expression of heat shock proteins such as SSA4, the cell shuts off the export of most transcripts 42° C, but the SSA4 transcript is selectively exported. The method by which SSA4 is able to export under these conditions is not understood. Previous attempts in our lab have aimed at using a reporter to allow visualization of SSA4 expression and export. Specifically, this reporter includes the green fluorescent protein (GFP) open reading frame with SSA4 regulatory sequence. However, this reporter does not recapitulate the expression of previously-reported endogenous SSA4. Therefore, the goal of this study is to evaluate the expression kinetics of the exogenous SSA4-GFP reporter in comparison to endogenous SSA4. We hypothesize that exogenous and endogenous SSA4 are induced similarly, but that the reporter transcript is not able to exit the nucleus. To explore the kinetics of induction we have first established a Q-PCR system that displays induction of endogenous SSA4 in response to heat shock. Ongoing experiments are aimed at Q-PCR analysis of exogenous SSA4-GFP in comparison to endogenous SSA4.
Keywords: S. cerevisiae, SSA4, nuclear pore complex, heat shock, GFP, chaperones, mRNA, selective export, gene expression, qPCR
Sharanshi, Susveen and Adams, Rebecca, "Analysis of SSA4 Reporter Expression by Q-PCR" (2023). Belmont University Research Symposium (BURS). 192.