Abstract
RNA-binding proteins (RBPs) establish the cellular fate of a transcript, but an understanding of these processes has been limited by a lack of identified specific interactions between RNA and protein molecules. Using MS2 RNA tagging, we have purified proteins associated with individual mRNA species induced by osmotic stress, STL1 and GPD1. We found members of the Lsm1-7/Pat1 RBP complex to preferentially bind these mRNAs, relative to the non-stress induced mRNAs, HYP2 and ASH1. To assess the functional importance, we mutated components of the Lsm1-7/Pat1 RBP complex and analyzed the impact on expression of osmostress gene products. We observed a defect in global translation inhibition under osmotic stress in pat1 and lsm1 mutants, which correlated with an abnormally high association of both non-stress and stress-induced mRNAs to translationally active polysomes. Additionally, for stress-induced proteins normally triggered only by moderate or high osmostress, in the mutants the protein levels rose high already at weak hyperosmosis. Analysis of ribosome passage on mRNAs through co-translational decay from the 5’ end (5P-Seq) showed increased ribosome accumulation in lsm1 and pat1 mutants upstream of the start codon. This effect was particularly strong for mRNAs induced under osmostress. Thus, our results indicate that, in addition to its role in degradation, the Lsm1-7/Pat1 complex acts as a selective translational repressor, having stronger effect over the translation initiation of heavily expressed mRNAs. Binding of the Lsm1-7/Pat1p complex to osmostress-induced mRNAs mitigates their translation, suppressing it in conditions of weak or no stress, and avoiding a hyperresponse when triggered.
Highlights
The regulation of the gene expression is essential to all cells; the proper protein accumulation is controlled at multiple steps, including transcription and translation, as well as mRNA and protein transport and stability
When confronted with external physical or chemical stress, cells respond by increasing the mRNA output of a small number of genes required for stress survival, while shutting
Our goal was to identify RNA-binding proteins (RBPs) involved in differential regulation of specific mRNAs under hyperosmotic stress
Summary
The regulation of the gene expression is essential to all cells; the proper protein accumulation is controlled at multiple steps, including transcription and translation, as well as mRNA and protein transport and stability. Post-transcriptional control may explain some 20% of steady-state mRNA levels, during rapidly changing conditions, post-transcriptional regulation is crucial for the initial responses [1]. For rapid adaptations to preserve energy, post-transcriptional regulation acting on pre-existing mRNAs plays a major role by virtue of being faster than changes on the level of transcription initiation, and by intervening before the costly step of protein synthesis [6]. RBPs are fundamental for regulating transcript stability, localization, and translational efficiency, and sorting which mRNAs are to be expressed into proteins under specific conditions, contributing to stress survival and recovery. The RBP Slf promotes translation of a few mRNAs during peroxide stress through interaction with the ribosome; slf mutants are hypersensitive to oxidative stress [10]
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