Sciences and Mathematics, College of
Biology, Department of
BURS Faculty Advisor
Rebecca Adams, PhD
Yeast two-hybrid (Y2H) to test Dbp5-Gle1 interaction dependence on Nup42
Gia Gia Le and Rebecca Adams, PhD
For proteins to be made in eukaryotes, mature mRNA must be exported from the nucleus to reach the cytoplasm, the site of translation. Mex67, a protein that binds the transcript in the nucleus, binds to nuclear pore complex (NPC) proteins allowing mRNAs to travel through the selective NPC into the cytoplasm. Mex67 is then removed by a protein called Dbp5 to provide directional export of the mRNA. Dbp5 activity is regulated by Gle1, and recent experiments have suggested that the Dbp5-Gle1 interaction is dependent on a NPC protein called Nup42. My project is to assess which domain of Nup42, either the full-length or a deletion termed nup42ΔFG is sufficient for the Dbp5-Gle1 interaction. This interaction can be observed through a process called yeast two-hybrid (Y2H) assay. In short, a Y2H assay is a genetic approach that assesses protein-protein interactions by the resulting phenotype in engineered cells: cells are only viable if the two proteins interact, and cells die in these conditions if proteins don’t interact. Thus far in this project, I have engineered a GAD-DBP5 Y2H plasmid from a LEU2 to a URA3 plasmid, by using methods of Phusion PCR, Gibson Cloning, bacterial transformation, and colony PCR. Next, I will transform this plasmid and GBD-GLE1 into wild-type and nup42Δ Y2H reporter strains. Following this, full-length NUP42 or nup42ΔFG plasmids will be transformed to test for viability. Using the information obtained from this study, we can begin to have a complete picture of the interactions mediating the mRNA export process.
Le, Gia and Adams, Rebecca, "Yeast two-hybrid (Y2H) to test Dbp5-Gle1 interaction dependence on Nup42" (2022). Belmont University Research Symposium (BURS). 75.