Effect of Arginine Methylation on Ded1 Function in Saccharomyces cerevisiae

Abstract

Ded1p is an ATP‐dependent RNA helicase that promotes translation and accumulates in stress granules. Ded1 is essential in budding yeast, promotes the translation of a large number of mRNA transcripts, and cooperates with eIF4G and eIF4A to promote translation initiation (Gao et al., 2016; Sen et al., 2015; Berthelot, 2005). We are interested in how Ded1p function is regulated via post‐translational modifications. Ded1p is methylated by Hmt1 at four arginines–three in the N terminus and one in the C terminus (Erce et al., 2013). We generated mutant alleles that alter these four amino acids to alanine (ded1‐quadA) or lysine (ded1‐quadK). Alanine cannot be methylated, is smaller and neutral, while lysine would similarly not be methylated, but has the same charge and similar shape as arginine. These ded1 alleles can complement a ded1 null, indicating that the residues, and their methylation, are not required for the essential function of DED1.To test whether these ded1 mutants are impaired in translation, we focused on highly structured 5′ UTRs from natural transcripts that have been shown to require Ded1p; these 5′ UTRs are fused to a luciferase reporter and expressed in yeast (Sen et al., 2015). We tested four reporters and identified one transcript, with the CLG1 5′ UTR, that is poorly translated in yeast containing ded1‐quadA. These results suggest that these Ded1 residues, or their methylation, promote the translation of this transcript. We want to determine whether the translation of other mRNAs is sensitive to Ded1 methylation mutants. We have isolated polysomes by sucrose gradient and will identify the translating mRNAs by RNA‐seq to determine which transcripts are depleted from polysomes in ded1‐quadA cells.Ded1 interacts with the nuclear export factor Npl3; this interaction is strengthened when both proteins are methylated by Hmt1 (Erce et al., 2013). We are testing whether methylation of Ded1 affects its known interactions with translation initiation factors. Ded1 physically interacts and cooperates with eIF4A (Gao et al., 2012). The human ortholog of Ded1, DDX3, has been shown to physically interact with eIF4E, the cap binding protein (Shih et al., 2008). Additionally, Ded1 has been shown to directly interact with eIF4G (Hilliker et al., 2011) and itself (Putnam et al., 2015). As these interactions are achieved via the N or C termini of Ded1, methylation of Ded1 could affect the binding of Ded1 with these factors. To determine whether methylation affects Ded1's interactions, we are using a conditional two‐hybrid assay in E. coli (Erce et al., 2013). Ded1 will be introduced as bait and a binding partner as prey; two‐hybrid interactions will be measured in the presence or absence of Hmt1 co‐expression to assess the importance of methylation on the interaction. Through these assays, we will explore whether methylation of Ded1 affects its role in translation initiation.Support or Funding InformationJeffress Memorial Trust Program in Interdisciplinary ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.