Quantification of elongation stalls and impact on gene expression in yeast.

Ribosomal pauses are a critical part of cotranslational events including protein folding and localization. However, extended ribosome pauses can lead to ribosome collisions, resulting in the activation of ribosome rescue pathways and turnover of protein and mRNA. While this relationship has been known, there has been little exploration of how ribosomal stalls impact translation duration at a quantitative level. We have taken a method used to measure elongation time and adapted it for use in Saccharomyces cerevisiae to quantify the impact of elongation stalls. We find, in transcripts containing Arg CGA codon repeat-induced stalls, a Hel2-mediated dose-dependent decrease in protein expression and mRNA level and an elongation delay on the order of minutes. In transcripts that contain synonymous substitutions to nonoptimal Leu codons, there is a decrease in protein and mRNA levels, as well as similar elongation delay, but this occurs through a non-Hel2-mediated mechanism. Finally, we find that Dhh1 selectively increases protein expression, mRNA level, and elongation rate. This indicates that distinct poorly translated mRNAs will activate different rescue pathways despite similar elongation stall durations. Taken together, these results provide new quantitative mechanistic insight into the surveillance of translation and the roles of Hel2 and Dhh1 in mediating ribosome pausing events.

Quantification of elongation stalls and impact on gene expression in yeast.

Ribosomal pauses are a critical part of co-translational events including protein folding and localization. However, extended ribosome pauses can lead to ribosome collisions, resulting in the activation of ribosome rescue pathways and turnover of protein and mRNA. While this relationship has been known, the specific threshold between permissible pausing versus activation of rescue pathways has not been quantified. We have taken a method used to measure elongation time and adapted it for use in S. cerevisiae to quantify the impact of elongation stalls. We find, in transcripts containing Arg CGA codon repeat-induced stalls, a Hel2-mediated dose-dependent decrease in protein expression and mRNA level and an elongation delay on the order of minutes. In transcripts that contain synonymous substitutions to non-optimal Leu codons, there is a decrease in protein and mRNA levels, as well as similar elongation delay, but this occurs through a non-Hel2-mediated mechanism. Finally, we find that Dhh1 selectively increases protein expression, mRNA level, and elongation rate. This indicates that distinct poorly translated codons in an mRNA will activate different rescue pathways despite similar elongation stall durations. Taken together, these results provide new quantitative mechanistic insight into the surveillance of translation and the roles of Hel2 and Dhh1 in mediating ribosome pausing events.

Rvb1/Rvb2 proteins couple transcription and translation during glucose starvation.

During times of unpredictable stress, organisms must adapt their gene expression to maximize survival. Along with changes in transcription, one conserved means of gene regulation during conditions that quickly repress translation is the formation of cytoplasmic phase-separated mRNP granules such as P-bodies and stress granules. Previously, we identified that distinct steps in gene expression can be coupled during glucose starvation as promoter sequences in the nucleus are able to direct the subcellular localization and translatability of mRNAs in the cytosol. Here, we report that Rvb1 and Rvb2, conserved ATPase proteins implicated as protein assembly chaperones and chromatin remodelers, were enriched at the promoters and mRNAs of genes involved in alternative glucose metabolism pathways that we previously found to be transcriptionally upregulated but translationally downregulated during glucose starvation in yeast. Engineered Rvb1/Rvb2-binding on mRNAs was sufficient to sequester mRNAs into mRNP granules and repress their translation. Additionally, this Rvb tethering to the mRNA drove further transcriptional upregulation of the target genes. Further, we found that depletion of Rvb2 caused decreased alternative glucose metabolism gene mRNA induction, but upregulation of protein synthesis during glucose starvation. Overall, our results point to Rvb1/Rvb2 coupling transcription, mRNA granular localization, and translatability of mRNAs during glucose starvation. This Rvb-mediated rapid gene regulation could potentially serve as an efficient recovery plan for cells after stress removal.

DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection.

Several arenaviruses cause hemorrhagic fever (HF) diseases that are associated with high morbidity and mortality in humans. Accordingly, HF arenaviruses have been listed as top-priority emerging diseases for which countermeasures are urgently needed. Because arenavirus nucleoprotein (NP) plays critical roles in both virus multiplication and immune-evasion, we used an unbiased proteomic approach to identify NP-interacting proteins in human cells. DDX3, a DEAD-box ATP-dependent-RNA-helicase, interacted with NP in both NP-transfected and virus-infected cells. Importantly, DDX3 deficiency compromised the propagation of both Old and New World arenaviruses, including the HF arenaviruses Lassa and Junin viruses. The DDX3 role in promoting arenavirus multiplication associated with both a previously un-recognized DDX3 inhibitory role in type I interferon production in arenavirus infected cells and a positive DDX3 effect on arenavirus RNA synthesis that was dependent on its ATPase and Helicase activities. Our results uncover novel mechanisms used by arenaviruses to exploit the host machinery and subvert immunity, singling out DDX3 as a potential host target for developing new therapies against highly pathogenic arenaviruses.