Making an SSA4-ADE3 Reporter for Multicopy Suppression Screen in S. Cerevisiae
Gene expression is essential to life and occurs through the central dogma, a process that describes how DNA becomes proteins. Using the budding yeast S. cerevisiae as a model system, we can examine mRNA export through nuclear pore complexes (NPCs) to the cytoplasm to better understand the regulation of steps within the flow of genetic information. Individual mRNA transcripts exit the nucleus using Mex67 which bind mRNA via adaptor proteins and allow crossing through NPCs. However, during heat shock (42C) known adaptor proteins are rendered dysfunctional thus halting mRNA export. Under these conditions, the chaperone-encoding SSA4 mRNA is able to export the nucleus and be translated, but the mechanism for this selective export is not understood.
I hypothesize that an unknown adaptor protein recruits Mex67 to allow for its selective export from the nucleus. In order to test this hypothesis, I have cloned a vector to be used later as phenotypic reporters for SSA4 export using the ADE3 ORF which yields a red yeast cell if SSA4 is correctly exported during heat shock conditions. I have successfully generated a UbiY-ADE3 plasmid featuring the SSA4 5’ and 3’ UTR regions. Future analysis will test whether this reporter successfully recapitulates selective SSA4 expression and export.
Leave a Reply