Stress-induced translation inhibition through rapid displacement of scanning initiation factors

Abstract

Running title: Stress-induced changes in the RNA-bound proteome Highlights:A quantitative proteomic approach reveals rapid stress-induced changes in RNA-binding Translation shutdown is driven by loss of mRNA binding by scanning initiation factors eIF4B and Ded1 have key but separate roles in driving the stress response Heat shock invokes rapid RNA degradation by Xrn1, selective for translation machinery 2 SUMMARY Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome, without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNAassociation. Among translation components, RNA-association was most reduced for initiation factors involved in 40S scanning (eIF4A, eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5′ends of mRNAs. Following glucose withdrawal, 5'-binding was abolished within 30sec, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5' RNA-binding by initiation factors over ~16min. Translation shutoff provoked selective 5′-degradation of mRNAs encoding translation-related factors, mediated by Xrn1. These results reveal mechanisms underlying translational control of gene expression during stress.

Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition interface

“…S4C). We compared the CRAC reads to poly(A)-enriched RNA-seq from the background (untagged) yeast strain grown in matched conditions (Bresson 2020), which were also reproducible ( Fig S4D). Counting reads on full-length transcripts, and normalising by transcript length and total density to transcripts per million (TPM) (Wagner, Kin, and Lynch 2012), revealed a clear enrichment for a small proportion of mRNAs ( Fig 4C).…”

“…CRAC studies were done in biological duplicates on cells growing exponentially in synthetic medium at 30°C, and following heat shock at 42°C for 16 minutes, a condition in which total Ssd1 binding to RNA markedly increases (Bresson 2020). Expression of tagged constructs was verified by western blot (Fig.…”

Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism

“…Using pcDNA5-FRT/TO as a starting point, we generated two additional parental vectors with pre-inserted tandem-affinity purification tags, either an N-terminal, FH-tag (FLAG-Ala4-His8) or Cterminal HF-tag (His8-Ala4-FLAG) ( Figure 2A). We have recently shown that these tags work well for tandem affinity purification, including in the denaturing conditions used for CRAC (Bresson et al, 2020).…”

“…Approximately 30 μg of protein was resolved on a 4-20% Miniprotean TGX gel, run in Tris-Glycine running buffer at 100 V. Subsequently, the gel was rinsed with water, stained for 1 hour with Imperial Protein Stain (Thermo Scientific), rinsed several times, and destained in water for three hours. Each lane was cut into four fractions and processed using in-gel digestion and the STAGE tip method, as previously described (Bresson et al, 2020;Rappsilber et al, 2007).…”

“…Our lab has previously developed techniques to map the RNA-bound proteome, including crosslinking and analysis of cDNA (CRAC), to identify binding sites for proteins on RNA, and total RNA-associated proteome purification (TRAPP) to identify and quantify the RNA-bound proteome (Granneman et al, 2009;Shchepachev et al, 2019). Among other things, TRAPP has given insights into the mechanism of stress-induced translation shutdown in yeast (Bresson et al, 2020). In the context of SARS-CoV-2, CRAC is expected to identify host RNAs that are targeted by viral factors, while TRAPP will reveal how host cell RNA-protein interactions are globally remodeled in response to specific viral proteins.…”