Impact of Mutations in the DEXD‐motif of the Yeast RNA‐dependent ATPase Sub2

Abstract

Pre‐mRNA splicing by the spliceosome is an essential part of eukaryotic gene expression. Errors in splicing can be detrimental to an organism, which is why it is extremely important to understand the spliceosome's mechanisms of assembly and action. Key features of splicing include the numerous RNA‐protein remodeling and RNA unwinding reactions that aid in spliceosome assembly. Some of these reactions require one of three, well‐conserved DEAD‐motif proteins that are part of the splicing machinery (Prp5/DDX46, Prp28/DDX23, and Sub2/UAP56). While Prp5 and Prp28 contain the canonical DEAD tetrapeptide motif within their active site, Sub2 is unusual in that it instead contains a conserved DECD motif. The goal of my research is to understand the function of the cysteine within Sub2's DECD motif in vitro and in vivo. Contrary to previous reports, I have shown that yeast containing a DECD to DEAD mutation in Sub2 are viable with little impact on yeast proliferation. To explain the conservation of Sub2's DECD sequence, we looked for changes in yeast phenotypes under stress conditions. I exposed yeast containing either DEAD‐Sub2 or DECD‐Sub2 to hydrogen peroxide and found that yeast containing DECD‐Sub2 died at lower oxidative stress conditions than the DEAD mutant. This suggests that Sub2's DECD‐motif cysteine is capable of being oxidized and that this results in a disruption in gene expression. Since one cysteine oxidation product (sulfinic acid) is isosteric with aspartate, I am now testing a DEDD mutant of Sub2 for function as well as attempting to chemically characterize the amino acid modifications resulting from Sub2 oxidation. Together my results will provide insight into a potential function for Sub2's conserved DECD‐motif cysteine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.