Regulation of translation initiation under stress conditions

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Two major steps of gene expression are transcription and translation. In the last 20 years, gene expression research has mainly focused on transcriptional regulation as microarray analysis became established. However, recent studies using genomic and proteomic profiling have revealed that changes in mRNA levels only partially correspond to those in protein levels. This suggests that translational regulation also plays critical roles in the regulation of gene expression. Translational regulation may be particularly important when cells respond to a rapid environmental change caused by cellular stresses. During the previous NSERC funding period, we investigated translation initiated by a unique RNA element in the 5'UTR of mRNA called the internal ribosomal entry site (IRES) under normal and stress conditions using a bicistronic reporter system. We determined that cellular stresses, such as amino acid starvation and mild hypothermia, increase the translation efficiency initiated by certain IRES elements. While the bicistronic reporter system has been very useful in these studies, it is not suitable for investigating a large number of IRES elements. Moreover, emerging evidence indicates that cis-acting elements other than IRES elements in the 5' and 3' UTR of mRNA can also regulate translation under stress conditions. Therefore, to fully understand how cellular stresses induce changes in translatomes, a global analysis is needed. To this end, we propose to investigate cellular stress-induced changes in the translatome using polysome profiling combined with RNA sequencing. The goal is to identify proteins involved in cell survival and death that are translationally regulated by cis-elements on the 5' and 3'UTR of mRNA under cellular stresses. Objective 1: Identification of proteins translationally regulated under stress conditions. mRNA preparations will be fractionated based on the number of bound ribosomes by conducting polysome profiling. mRNAs isolated from heavy polysome fractions (actively translated) of cells under normal and stress conditions will be compared by RNA sequencing. Objective 2: Validation of polysome profiling/RNA sequencing data. The results obtained in Objective 1 will be confirmed by western blot analysis, quantitative RT-PCR, de novo protein synthesis assay and 5' or 3'UTR reporter analysis. Objective 3: Investigation of functions of the validated proteins under stress conditions. Many of the proteins translationally upregulated during cellular stress are most likely very important for regulating cell survival and death. The functions of the proteins validated in Objective 2 will be determined by loss- and gain-of-function experiments.To our knowledge, there are very few laboratories conducting a genome-wide scale analysis on translatome of cells under stress conditions. Thus, the proposed research program represents a new frontier.