Genome-wide discovery of translational control mechanisms.

Abstract

Genome-wide discovery of translation control mechanisms

The control of mRNA translation into proteins is critical for the adaptation of eukaryotic cells to environmental changes and stress conditions. Glucose starvation in yeast is one of the prototypical eukaryotic stresses. The early translation-based responses to glucose starvation are critically important to trigger the subsequent events leading to transcriptional reprogramming, but the mRNAs involved and the mechanisms of their selective regulation remain obscure. I analysed glucose-specific translational control primarily using Translation Complex Profile sequencing (TCP-seq). TCP-seq measures the distribution of ribosomal complexes along mRNA using a combination of in vivo fixation, complex purification and limited nuclease digestion, and high-throughput sequencing of the protected mRNA fragments (footprints). Here, to dissect different aspects of translation regulation, I separately recorded footprints of single ribosomes and disomes. I furthermore sequenced footprints of three distinct complexes involving small ribosomal subunits (SSU), those derived from polysomes, 'singular' subunits in solution, as well as those that selectively contained the scanning translation initiation factor eIF4A. During starvation, total RNA-seq revealed transcripts involved in metabolic processes are downregulated whereas transcripts involved in alcoholic catabolic processes are upregulated, as a coping mechanism to economize their energy and shift to alcohol as a source of nutrition after longer duration of starvation. Promoter analysis of these clusters revealed the presence of motifs that are indicative of modulating expression of ribosomal protein genes and ribosome biogenesis genes that are consistent with links to cell growth, stress and nutrient conditions. Investigating different cellular pools with RNA-seq hints towards rapid sequestration into granules as well. Under starvation and globally suppressed protein biosynthesis, I observed an increased level of translation of many glucosynthetic mRNAs, mRNAs encoding heat shock proteins, hexokinase, 3-phosphoglycerate kinase. We also demonstrate that stochastically co-localised ribosomes are linked with translation imitation rate and provide a robust variable to model and quantify specific protein output from mRNA. I further propose a new measure of translation efficiency (TE) which may be more robust to the regularly-encountered biases of the classical TE measure and can contain more accurate information, enabling ranking mRNAs by the absolute protein output during rapidly changing transcriptome background, as occurs in glucose starvation. Overall, these data uncover a complex picture of rapid translational changes and present a collection of transcripts involved in the primary, acute response to the stress. The results suggest that already early in the response several major pathways are involved in the translational control simultaneously, as mRNAs are specifically degraded or preserved and their translation is selectively shut down, unperturbed or upregulated.