Abstract
The mechanisms by which RNA-binding proteins control the translation of subsets of mRNAs are not yet clear. Slf1p and Sro9p are atypical-La motif containing proteins which are members of a superfamily of RNA-binding proteins conserved in eukaryotes. RIP-Seq analysis of these two yeast proteins identified overlapping and distinct sets of mRNA targets, including highly translated mRNAs such as those encoding ribosomal proteins. In paralell, transcriptome analysis of slf1Δ and sro9Δ mutant strains indicated altered gene expression in similar functional classes of mRNAs following loss of each factor. The loss of SLF1 had a greater impact on the transcriptome, and in particular, revealed changes in genes involved in the oxidative stress response. slf1Δ cells are more sensitive to oxidants and RIP-Seq analysis of oxidatively stressed cells enriched Slf1p targets encoding antioxidants and other proteins required for oxidant tolerance. To quantify these effects at the protein level, we used label-free mass spectrometry to compare the proteomes of wild-type and slf1Δ strains following oxidative stress. This analysis identified several proteins which are normally induced in response to hydrogen peroxide, but where this increase is attenuated in the slf1Δ mutant. Importantly, a significant number of the mRNAs encoding these targets were also identified as Slf1p-mRNA targets. We show that Slf1p remains associated with the few translating ribosomes following hydrogen peroxide stress and that Slf1p co-immunoprecipitates ribosomes and members of the eIF4E/eIF4G/Pab1p ‘closed loop’ complex suggesting that Slf1p interacts with actively translated mRNAs following stress. Finally, mutational analysis of SLF1 revealed a novel ribosome interacting domain in Slf1p, independent of its RNA binding La-motif. Together, our results indicate that Slf1p mediates a translational response to oxidative stress via mRNA-specific translational control.
Highlights
The control of translation in response to external stimuli plays an important role in the regulation of gene expression
We show that a previously identified RNA-binding protein called Slf1p plays a major role in mRNA-specific regulation of translation during oxidative stress conditions and is necessary to promote the translation of stress-responsive mRNAs
Exposure to oxidants is known to cause a general down-regulation of protein synthesis, many stress response proteins are able to overcome this inhibition and increase their protein levels following stress by as yet unknown mechanisms
Summary
The control of translation in response to external stimuli plays an important role in the regulation of gene expression. Some estimates of the relative contributions of different molecular mechanisms to the overall control of gene expression highlight a dominant role for translational control [1,2]. In response to external stimuli, such as amino acid starvation or hydrogen peroxide stress, global translation initiation is normally reduced whilst significant numbers of specific mRNAs continue to be translated [3]. In addition to intrinsic mRNA properties, a large number of RNA binding proteins (RBPs) are known to bind specific mRNAs in order to either activate or repress their translation [4], forming a cellular network of post-transcriptional regulation above that exerted at the transcriptional level
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