Yeast Three Hybrid Screen in Search of SSA4 3’ UTR Binding Protein in S. cerevisiae
Sciences and Mathematics, College of
Biology, Department of
BURS Faculty Advisor
Rebecca Adams, PhD
In eukaryotic cells, gene expression requires the exit of mature mRNA transcripts from the nucleus, where they are generated, to the cytoplasm, where they are decoded into protein. During this process, adaptor proteins bind to transcripts to regulate whether they can travel through nuclear pore complexes, selective doorways embedded within the nuclear envelope. However, when the cell is in a stressful environment, such as heat shock, the export of most mRNA is halted, and known adaptors are rendered non-functional. This allows the cell to respond by inducing expression of stress-responsive genes. Select RNA transcripts, such as SSA4, are permitted to export out of the nucleus under these stress conditions, allowing for their specific expression. Previous studies have indicated that the 3’UTR of SSA4 mediates this selective export. The goal of my project is to identify the mechanism of selective mRNA export via the SSA4 3’UTR. I hypothesized that an unknown adaptor protein binds to the SSA4 3’UTR to allow for export. To identify the adaptor protein, I have employed yeast three-hybrid (Y3H) screening, which is a genetic approach to detect RNA-protein interactions. I performed a large-scale Y3H genetic screen to identify the unknown protein that possibly interacts with the SSA4 3’UTR. Currently, I have identified 5 hits that indicate a positive Y3H via two phenotypic outcomes. To confirm specificity, I will analyze interactions following loss of the SSA4 3’UTR reporter and with a negative control. Additionally, I will isolate the plasmids from hits to sequence the DNA to identify the unknown adaptor protein interacting with SSA4 3’ UTR. We anticipate that this will allow identification of the hypothesized SSA4 3’UTR binding protein. Further, identifying this mechanism will enable broader understanding of how cells enable drastic changes to gene expression in response to environmental stimuli.
Irbe, Emma, "Yeast Three Hybrid Screen in Search of SSA4 3’ UTR Binding Protein in S. cerevisiae" (2022). Belmont University Research Symposium (BURS). 123.