Assessing Requirement of Dbp5 ATPase Activity for Interaction with Nab2
In Saccharomyces cerevisiae, mature mRNA exits the nucleus following transcription to permit translation in the cytoplasm in a process called mRNA export. During this process, 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. To prohibit mRNA entry back into the nucleus, the protein Dbp5 removes these proteins from the transcript through ATPase activity, allowing Nab2 and Mex67 to recycle back into the nucleus. It is unknown how Dbp5 specifically removes Nab2 and not other RBPs that bind to the transcript in the nucleus and are maintained in the cytoplasm. I hypothesize that Dbp5 binds to Nab2, and this brings Dbp5 to the region of the transcript where they bind for removal. This interaction can be observed using a Split-Venus test. My goal is to assess whether Dbp5 activity is required for the Dbp5-Nab2 interaction. To do this, I cloned mutations of Dbp5 that disrupt its activity (ATP binding, RNA binding, and ATP Hydrolysis) into a galactose-inducible Split-Venus plasmid. I then tested for Split-Venus interaction with Nab2 through fluorescence microscopy. The resulting yeast strains demonstrated enriched fluorescence in mutations for RNA binding while mutations for ATP binding and ATPase had a less distinct signal. These preliminary results indicate that RNA binding of Dbp5 is not essential for interaction with Nab2, therefore providing more details about this interaction. In future experiments, the Split-Venus signal should be quantified to determine the significance of the observed fluorescence.
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