Abstract 1902 The Role of Arginine Methylation of the RNA Helicase, Ded1, in Promoting Translation

Ded1p is an essential RNA helicase that promotes nuclear export (1), stress granule accumulation, and translation initiation. Ded1 interacts with a number of translation initiation factors, including eIF4A, eIF4E, and eIF4G (2, 3). Ded1 is methylated by Hmt1 at five arginine residues within its intrinsically disordered domains (4, 5). Methylation strengthens the interaction between Ded1 and Npl3, a protein involved in nuclear export (4), but it is unclear if methylation impacts Ded1's functions in translation or stress granule accumulation. Ded1 is methylated under a range of growth conditions and stresses, suggesting this modification is common in vivo. To test the function of Ded1 methylation, we mutated four methylated arginines to either alanine (ded1-quadA) or lysine (ded1-quadK). These mutants show no detectable methylation by western blot, indicating that methylation is significantly decreased. These mutants complement a null and show minimal effect on polysome traces, demonstrating that Ded1 methylation is not required for global translation. However, Ded1 methylation may be important for a subset of mRNAs. Consistent with this idea, the methylation deficient mutants show a range of phenotypes distinct from wild type yeast, which were identified via phenotypic microarray screen. These phenotypes suggest that the lack of methylation becomes important under certain growth conditions. Additionally, the methylation defective ded1 mutant fails to localize to stress granules. Ded1 is important for the translation of many mRNAs, especially those with highly structured 5' UTRs (6). We have screened several Ded1-dependent translation reporters for their translation efficiency in cells with methylation defective ded1 mutants. We found one reporter that is sensitive to the methylation defective mutant and have assessed the entire transcriptome for mRNAs that are similarly affected by Ded1 methylation. We are currently analyzing these results to identify any similarities in GO terms, structure potential, length, etc. among the RNAs whose translation is sensitive to the methylation defective ded1 mutant. We have successfully purified recombinant methylated versus non-methylated Ded1. We see no significant difference in efficiency of in vitro helicase activity in methylated versus unmethylated rDed1. We are currently testing whether methylation affects other biochemical functions of Ded1, such as its ability to interact with known or hypothesized binding partners. For example, we find new evidence that Ded1 interacts with Dbp1, a paralog of Ded1 in yeast, but this interaction is not sensitive to methylation. In summary, methylation of Ded1 is important for growth in specific nutrient conditions and important for a subset of mRNAs. Additionally, it is important for Ded1's accumulation in stress granules. While methylation does not impact Ded1's intrinsic helicase activity, methylation occurs in areas of the protein that promote interaction with eIF4F. We are currently testing whether Ded1 methylation changes interactions with eIF4F or the extent to which eIF4F proteins can impact Ded1 helicase activity (2). (1) Hauk and Bowman. 2015. PloS One. 10: e0131690. (2) Gao et al. 2016. eLife 5:e16408. (3) Hilliker et al. 2011. Molecular Cell 43: 962-972. (4) Erce et al. 2013. Molecular & Cellular Proteomics 12: 3184-3198. (5) Hamey et al. 2021. J. Proteome Res. 20(5): 2420–2434. (6) Sen et al. 2015. Genome Res 25: 1196-205. A.K.H. was funded by the NIH (NIGMS R15-GM128068) and the University of Richmond School of Arts & Sciences. All co-authors are undergraduates funded by the same NIH grant and/or the University of Richmond School of Arts & Sciences.