Altering Conserved Residues on Dbp5 in S. cerevisiae to Assess Function
Sciences and Mathematics, College of
Biology, Department of
BURS Faculty Advisor
Nuclear mRNA export is essential for the viability of eukaryotic cells as it enables gene expression. This process allows for mRNA, the genetic copy of DNA that encodes for proteins, to be transported from the nucleus, where it is generated, to the cytoplasm for translation. The mRNA transcript requires several binding proteins to cross through nuclear pore complexes (NPCs), which are selective doorways embedded in the nuclear envelope. Ultimately, binding to Mex67 allows for the transcript to be transported through the NPC. Once the transcript reaches the cytoplasm, another protein called Dbp5 binds to the mRNA and removes Mex67, preventing the mRNA from re-entering the nucleus. Along with Mex67, several other proteins bind to mRNA in the nucleus but remain on the transcript in the cytoplasm. How Dbp5 selectively removes Mex67 and not other RNA binding partners is unknown. We hypothesize that Dbp5 might have an unknown binding partner, potentially Mex67, that enables this selectivity. Dbp5 has several binding partners, but we identified a patch of conserved amino acids on Dbp5 that is distinct from interfaces of known binding partners. This patch having been evolutionarily maintained between humans and yeast indicates that the site likely holds a significant function for cells, possibly an interface with Mex67. In order to test our hypothesis, I generated mutations using site-directed PCR mutagenesis. The mutated plasmids were then confirmed by Sangar sequencing and transformed into S. cerevisiae to assess the mutant Dbp5 functionality. As Dbp5 is essential for cell viability, Dbp5 mutants will result in inviable cells if this patch of conserved amino acids is important for mRNA export. We anticipate that a combination of mutations will reduce Dbp5 functionality, and subsequent experiments will then determine whether the Mex67 interface is disrupted in these mutants.
Ford, Maddy and Adams, Rebecca, "Altering Conserved Residues on Dbp5 in S. cerevisiae to Assess Function" (2023). Belmont University Research Symposium (BURS). 219.