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The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an ATP-dependent RNA-binding protein and an RNA-dependent ATPase, but it was previously found to lack substrate specificity and enzymatic regulation. Here we demonstrate through yeast genetics, yeast extract pull-down experiments, in situ localization, and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 in the presence of SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results provide a new understanding of the role of Ded1 during translation.

Preexposure to mild stress often improves cellular tolerance to subsequent severe stress. Severe ethanol stress (10% v/v) causes persistent and pronounced translation repression in Saccharomyces cerevisiae. However, it remains unclear whether preexposure to mild stress can mitigate translation repression in yeast cells under severe ethanol stress. We found that the translational activity of yeast cells pretreated with 6% (v/v) ethanol was initially significantly repressed under subsequent 10% ethanol but was then gradually restored even under severe ethanol stress. We also found that 10% ethanol caused the aggregation of Ded1, which plays a key role in translation initiation as a DEAD-box RNA helicase. Pretreatment with 6% ethanol led to the gradual disaggregation of Ded1 under subsequent 10% ethanol treatment in wild-type cells but not in fes1Δhsp104Δ cells, which are deficient in Hsp104 with significantly reduced capacity for Hsp70. Hsp104 and Hsp70 are key components of the bi-chaperone system that play a role in yeast protein quality control. fes1Δhsp104Δ cells did not restore translational activity under 10% ethanol, even after pretreatment with 6% ethanol. These results indicate that the regeneration of Ded1 through the bi-chaperone system leads to the gradual restoration of translational activity under continuous severe stress. This study provides new insights into the acquired tolerance of yeast cells to severe ethanol stress and the resilience of their translational activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Macroautophagy/autophagy is a highly conserved nutrient-recycling pathway that eukaryotes utilize to combat diverse stresses including nutrient depletion. Dysregulation of autophagy disrupts cellular homeostasis leading to starvation susceptibility in yeast and disease development in humans. In yeast, the robust autophagy response to starvation is controlled by the upregulation of ATG genes, via regulatory processes involving multiple levels of gene expression. Despite the identification of several regulators through genetic studies, the predominant mechanism of regulation modulating the autophagy response to subtle differences in nutrient status remains undefined. Here, we report the unexpected finding that subtle changes in nutrient availability can cause large differences in autophagy flux, governed by hitherto unknown post-transcriptional regulatory mechanisms affecting the expression of the key autophagyinducing kinase Atg1 (ULK1/ULK2 in mammals). We have identified two novel post-transcriptional regulators of ATG1 expression, the kinase Rad53 and the RNA-binding protein Ded1 (DDX3 in mammals). Furthermore, we show that DDX3 regulates ULK1 expression post-transcriptionally, establishing mechanistic conservation and highlighting the power of yeast biology in uncovering regulatory mechanisms that can inform therapeutic approaches.

DEAD-box RNA helicase Ded1 is thought to resolve secondary structures in mRNA 5'-untranslated regions (5'-UTRs) that impede 48S preinitiation complex (PIC) formation at the initiation codon. We reconstituted Ded1 acceleration of 48S PIC assembly on native mRNAs in a pure system, and recapitulated increased Ded1-dependence of mRNAs that are Ded1-hyperdependent in vivo. Stem-loop (SL) structures in 5'-UTRs of native and synthetic mRNAs increased the Ded1 requirement to overcome their intrinsically low rates of 48S PIC recruitment. Ded1 acceleration of 48S assembly was greater in the presence of eIF4F, and domains mediating one or more Ded1 interactions with eIF4G or helicase eIF4A were required for efficient recruitment of all mRNAs; however, the relative importance of particular Ded1 and eIF4G domains were distinct for each mRNA. Our results account for the Ded1 hyper-dependence of mRNAs with structure-prone 5'-UTRs, and implicate an eIF4E·eIF4G·eIF4A·Ded1 complex in accelerating 48S PIC assembly on native mRNAs., Competing Interests: NG, JL No competing interests declared, AH Reviewing editor, eLife

In eukaryotic organisms, the orthologs of the DEAD-box RNA helicase Ded1p from yeast and DDX3 from human form a well-defined subfamily that is characterized by high sequence conservation in their helicase core and their N- and C- termini. Individual members of this Ded1/DDX3 subfamily perform multiple functions in RNA metabolism in both nucleus and cytoplasm. Ded1/DDX3 subfamily members have also been implicated in cellular signaling pathways and are targeted by diverse viruses. In this review, we discuss the considerable body of work on the biochemistry and biology of these proteins, including the recently discovered link of human DDX3 to tumorigenesis.