Belmont University Research Symposium (BURS)

Mutagenesis of conserved residues on Dbp5 structure and analysis of interaction with Nab2

Publication Date

Spring 3-29-2024

College

Sciences and Mathematics, College of

Department

Biology, Department of

BURS Faculty Advisor

Rebecca Adams

Presentation Type

Poster Presentation

Abstract

mRNA export is the process of mRNA transportation from the nucleus, where it is generated, to the cytoplasm for translation into proteins. For this to occur, the mRNA transcript requires a binding protein called Mex67 to allow for the passage through nuclear pore complexes (NPCs), selective doorways embedded in the nuclear envelope. Mex67 is recruited to mature transcripts via adaptor proteins, including Nab2, which signals the successful addition of a polyA tail. Once the mRNA transcript reaches the cytoplasm, Dbp5 detaches Mex67 and Nab2 from the mRNA to prevent the transcript from re-entering the nucleus. It is unknown how Dbp5 specifically removes Mex67 and Nab2 and none of the remaining RNA binding proteins. I hypothesize that Dbp5 binds to either Mex67 or Nab2, recruiting Dbp5 to the region of the transcript that allows for it to remove Mex67/Nab2 selectively. Indeed, previous studies in S. cerevisiae have observed an interaction between Dbp5 and Nab2 via split-GFP analysis: Dbp5-VC and Nab2-VN displayed a fluorescent signal at the nuclear envelope, indicating that they bind each other at the NPC. Additionally, it was observed that expression of Dbp5-VC in Nab2-VN mutants results in lethality, suggesting that Dbp5-VC functionally sequesters Nab2-VN from function. Further suggesting that Dbp5 has a novel binding partner, we have four evolutionary-conserved charged amino acid residues (K443, R463, D467, and E471) on the surface of Dbp5 away from known binding interfaces. To test whether these residues mediate interaction with Nab2, site-directed PCR mutagenesis was performed to introduce charge-reversal point mutations of these residues. Following the successful generation of mutants, these plasmids were transformed into yeast and plated on media containing galactose to induce expression of the Dbp5-VC mutant and assess cell viability. We observed that the D467K mutation had no significant effect on cell viability, suggesting that residue is not essential for the Dbp5-Nab2 interaction. Future experiments will be aimed at assessing the Dbp5-Nab2 interaction via fluorescent microscopy.

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