Investigating Translational Reprogramming In Cellular Stress Response By Elongation Pausing And Alternative Initiation
Cell survival in changing environments requires appropriate regulation of gene expression, including translational control. Multiple stress signaling pathways converge on several key translation factors and rapidly modulate mRNA translation at both the initiation and the elongation stages. Here, I discover that intracellular proteotoxic stress reduces global protein synthesis by halting ribosomes on transcripts during elongation. Deep sequencing of ribosome-protected mRNA fragments reveals an early elongation pausing, roughly at the site where nascent polypeptide chains emerge from the ribosomal exit tunnel. Inhibiting endogenous chaperone molecules by a dominant-negative mutant or chemical inhibitors recapitulates the early elongation pausing, suggesting a dual role of molecular chaperones in facilitating polypeptide elongation and co-translational folding. My results further support that trapped chaperone under stress may prevent the release of elongation factors from ribosomes. My study reveals that translating ribosomes fine-tune the elongation rate by sensing the intracellular folding environment. The early elongation pausing represents a co-translational stress response to maintain the intracellular protein homeostasis. Correspondingly, repression of global protein synthesis is often accompanied with selective translation of mRNAs encoding proteins that are vital for cell survival and stress recovery. Understanding the selective translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. Here, I develop an approach (global translation initiation sequencing, GTI-seq) to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. With single nucleotide resolution, I show an unprecedented view of alternative translation initiation in mammalian cells. Furthermore, I uncover a robust translational reprogramming of protein catabolic process, in particular the proteasome system, in response to starvation. This regulatory mode of TIS selection indicates that the scope of selective translation under stress conditions is much broader than anticipated. Collectively, my studies have revealed unprecedented proteome complexity and flexibility through stress-induced translational reprogramming, including ribosome pausing during elongation and wide-spread alternative translation initiation. Elucidation of the regulatory mechanisms underlying translational reprogramming will ultimately lead to the development of novel therapeutic strategies for human diseases.
Translation; Stress response; Ribosome profiling
Emr, Scott David; Cerione, Richard A
Ph.D. of Genetics
Doctor of Philosophy
dissertation or thesis