Methylation of Ded1 Affects Its Role in Translation

Abstract

Ded1p is an essential ATP‐dependent RNA helicase involved in translation initiation through association with translation factors such as eIF4A and eIF4G (1, 2), nuclear export and accumulation of stress granules. We are interested in how a post translational modification (PTM) of Ded1, methylation, modulates these roles of Ded1. The methyltransferase Hmt1 methylates Ded1 at four arginine residues in the N and C terminus (3). Methylation strengthens the interaction between Ded1 and Npl3, a protein involved in nuclear export (3). However, it is unclear how methylation impacts Ded1 in translation.To test Ded1 methylation in vivo, we mutated the four methylated arginines to either alanine or lysine. Methylation of Ded1p is not required for normal cell growth, making it unlikely that Ded1 methylation is required for global translation. However, Ded1 is important for the translation of many mRNAs, especially those with highly structured 5′ UTRs (8). We find a reporter with the highly structured CLG1 5′ UTR is not efficiently translated by a Ded1 methylation mutant, suggesting Ded1 methylation may be important for the translation of specific mRNAs.However, as the arginines are mutated, we cannot rule out another role for these arginines aside from methylation. In order to directly test the effect of Ded1 methylation on translation, we will purify recombinant wild type Ded1 protein with or without co‐expression with Hmt1. Translation efficiency of recombinant methylated versus unmethylated Ded1 protein will be tested in vitro using both simple and highly structured 5′ UTRs, such as CLG1, before a luciferase reporter. We will also perform helicase assays to test the impact of Ded1 methylation on its biochemical function.Since Ded1 interacts with other proteins through the N or C terminus, it is possible that methylation influences Ded1‐protein interactions. We use a conditional two‐hybrid assay in E. coli (3) to test the effect of methylation on Ded1's binding partners. We show methylation strengthens the known interaction between Ded1 and eIF4A. A novel interaction between Ded1 and Xrn1 and a reported oligomerization of Ded1 (4) has been validated through immunoprecipitation. Furthermore, we will test the effects of methylation on known Ded1 interactions with other translation factors such as eIF4G and eIF4E (2, 5) and RNA exporters such as Gle1 and XpoI (6, 7). Besides testing Ded1 interactions with a single binding partner, we will also test interactions between Ded1 and a combination of two different initiation factors. Ded1 is required for translation of >600 mRNAs in yeast (8). Our investigation further elucidates how methylation regulates Ded1 and its effect on mRNA translation.Support or Funding InformationNIH R15 GM128068‐01; HHMI; Arts & Sciences (UR); Smart FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.