Uncovering Dbp5 Residues Required for Interaction with Nab2
Sciences and Mathematics, College of
Biology, Department of
BURS Faculty Advisor
Rebecca Adams, Ph.D.
In Saccharomyces cerevisiae, as in all eukaryotes, mature mRNA exits the nucleus following transcription to permit translation in the cytoplasm in a process called mRNA export. To allow for transcript export, the RNA Binding Protein Nab2 binds to the poly-A tail and recruits RNA export protein Mex67 to allow the transcript to cross the nuclear pore complex and reach the cytoplasm for translation. To prohibit mRNA entry back into the nucleus, the enzyme Dbp5 removes Nab2 and Mex67 from the transcript through its ATPase activity, allowing their recycling back into the nucleus. It is unknown how Dbp5 specifically removes Nab2 and Mex67 and not the other RBPs that bind to the transcript in the nucleus and are maintained in the cytoplasm. I hypothesize that Dbp5 binds to Nab2 and/or Mex67, bringing Dbp5 to the region of the transcript where they bind for removal. Supporting this hypothesis, the interaction between Dbp5 and Nab2 can be observed using a split-Venus approach which not only displays fluorescence when Dbp5-VC and Nab2-VN are expressed but also results in cell death when Dbp5-VC is over-expressed in a Nab2-VN strain. My goal is to use the lethality that results from the split-Venus interaction to identify residues at the Dbp5-Nab2 interface. To do this, I performed random mutagenesis to generate a library of mutations within the Dbp5 ORF, of which some might prohibit the interaction between Dbp5 and Nab2. This library of mutants was then transformed into a Nab2-VN strain to uncover viable mutants, indicating a potentially disrupted interaction. These mutants will then be screened and sequenced to identify the mutation site. We anticipate that these results will aid in understanding Dbp5 selectivity.
Torres, Daisy, "Uncovering Dbp5 Residues Required for Interaction with Nab2" (2023). Belmont University Research Symposium (BURS). 217.