Sciences and Mathematics, College of
Biology, Department of
BURS Faculty Advisor
Dr. Rebecca Adams
The export of mature mRNA through the nuclear pore complex (NPC) is necessary for successful protein synthesis in the cytoplasm of eukaryotic cells. In S. cerevisiae, this process is directed by the conserved mRNA export protein Mex67, which ferries the transcript across the NPC. A protein called Dbp5 is responsible for removing Mex67 from the mRNA after it reaches the cytoplasm, providing directionality to the export process. In this role, Dbp5 is localized at the cytoplasmic side of the NPC through interaction with the NPC protein Nup159. Thus, the exiting RNA comes into contact with Dbp5 right after export for removal of Mex67. Dbp5 can also be found inside the nucleus and throughout the cytoplasm, where it has been implicated in the processes of transcription and translation, respectively, but the molecular function in these roles has not been defined. The goal of this study is to identify other functions of Dbp5 in subcellular localizations. I hypothesized that Dbp5 functions to remove other proteins from RNA in these other cellular compartments. In order to explore Dbp5 function and potentially uncover these putative Dbp5 targets, I have generated an integrated Dbp5-Nup159 strain in which Dbp5 is stably localized at the NPC. Thus, the nuclear and cytoplasmic Dbp5 functions have been lost, and this strain has a growth defect at increased temperatures (37°C). A previous student performed a multicopy suppressor screen to identify plasmids which allow growth at 37°C, possibly as a result of overexpression of target proteins of Dbp5. From these rescuing strains, I first utilized colony PCR to confirm that the rescued strains have a plasmid from the screen plasmid library, but lack endogenous Dbp5. Once positive strains were identified, the plasmids were isolated from the yeast, and transformed into E. coli to generate high concentration samples. We are currently test digesting the resulting plasmids followed by transformation into yeast to confirm the rescuing phenotype. The ultimate goal of this work is to sequence the plasmids in order to identify the rescuing gene. Once this is accomplished, identification of nuclear and cytoplasmic protein targets of Dbp5 is possible, allowing for a better understanding of the specific role of Dbp5 throughout the cell.
Harbison, Zoe, "Isolation of plasmids and confirmation of rescue from Dbp5-Nup159 multicopy suppressor screen" (2022). Belmont University Research Symposium (BURS). 125.