Ded1p is an essential ATP-dependent RNA helicase that promotes nuclear export (1), stress granule accumulation, and translation initiation through association with translation factors, such as eIF4A and eIF4G (2, 3). Ded1 is methylated by Hmt1 at five arginine residues in 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 translation. We find that Ded1 methylation is stably detected under a range of growth conditions and stresses, suggesting that this modification is common within in vivo stores of Ded1. To test the function of Ded1 methylation in vivo, we mutated four of the five methylated arginines to either alanine (ded1-quadA) or lysine (ded1-quadK). We are currently testing the effects of a recently discovered fifth methylation site (5). The ded1-quadA and quadK mutants show no Ded1 methylation detectable by western blot, indicating that methylation is significantly decreased. As these mutants complement a null, Ded1 methylation is not required for the essential function of Ded1 and is, therefore, unlikely to be required for global translation. Ded1 methylation may be important for a subset of mRNAs that are most dependent on Ded1 for translation (6). Consistent with this idea, the methylation deficient mutants do 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, but whether that affects stress granule formation or RNA accumulation within stress granules is unknown. 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. Recombinant methylated Ded1 is as efficient as non-methylated Ded1 in repressing translation in vitro when Ded1 is in excess, consistent with retaining aspects of Ded1 function. We are currently testing whether methylation affects other biochemical functions of Ded1, such as its ability to interact with eIF4A or eIF4G (2, 3). (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.

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Hilliker A, Cook A, Falco C

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