Site-specific K63 ubiquitinomics reveals post-initiation regulation of ribosomes under oxidative stress

Abstract

During oxidative stress, K63-linked polyubiquitin chains accumulate in the cell and modify a variety of proteins including ribosomes. Knowledge of the precise sites of K63 ubiquitin is key to understanding its function during the response to stress. To identify the sites of K63 ubiquitin, we developed a new mass-spectrometry based method that quantified >1,100 K63 ubiquitination sites in yeast responding to oxidative stress induced by H2O2. We determined that under stress, K63 ubiquitin modified proteins involved in several cellular functions including ion transport, protein trafficking, and translation. The most abundant ubiquitination sites localized to the head of the 40S subunit of the ribosome, modified assembled polysomes, and affected the binding of translation factors. The results suggested a new pathway of post-initiation control of translation during oxidative stress and illustrated the importance of high-resolution mapping of noncanonical ubiquitination events.

High-Resolution Ribosome Profiling Defines Discrete Ribosome Elongation States and Translational Regulation during Cellular Stress

“…Oxidative stress in S. cerevisiae has been shown to trigger multiple cellular responses that may impact translation elongation. For example, K63-linked polyubiquitination of several ribosomal proteins near the mRNA channel (Back et al, 2018;Silva et al, 2015) has been connected to oxidative stress and proposed to mediate elongation inhibition. Similarly, oxidative stress has been shown to stimulate tRNA cleavage in the anticodon region by Rny1, a member of the RNase T2 family (Thompson and Parker, 2009).…”