Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress

Abstract

Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5–3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4–2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.

Redox sensitive E2 Rad6 controls cellular response to oxidative stress via K63 ubiquitination of ribosomes

“…In the RTU, K63 ubiquitin is required to inhibit translation during cellular exposure to stress 3,4 . K63 ubiquitin modifies fully assembled 80S and polysomes and these complexes were mostly present in the pre-translocation stage of translation elongation, with a marked destabilization of the 60S P-stalk when compared to ribosomes from the K63R ubiquitin mutant strain 5 . Here we showed that RAD6 is required for global inhibition of translation during cellular exposure to H2O2 (Fig.…”

“…Furthermore, it has been shown that translation stalling and Gcn2 binding to proteins in the 60S P-stalk not engaged with elongation factors triggers the ISR 32,53,54 . Therefore, we propose that destabilization of the 60S P-stalk by K63 ubiquitin would provide a cross-talk mechanism between translation elongation and initiation under stress 5 . As a potential redox switch in the RTU, it will be also essential to understand the mechanisms of recruitment and activation of Gcn2 during oxidative stress as well as the fate of deubiquitinated ribosomes.…”

“…6). We also compared Ribo Rad6IP with the structure of K63 ubiquitinated ribosomes and ribosomes from the K63R ubiquitin mutant strain 5 . Although we saw the same extended 18S rRNA conformation in all the 3 datasets (Supplementary Fig.…”

Lies, Gosh Darn Lies, and not enough good statistics: why epidemic model parameter estimation fails

“…The rate of transmission depends on physiological parameters as well as normal social distance and social distancing response to an epidemic, how public institutions such as schools are run, how grocery shopping interactions are handled, whether known infections are isolated and other factors specific to each community. Given how widely these parameters may vary from population to population, and the mechanics of how they vary: how they depend on the geographically specific dominating SARS-COV-2 lineages dominant within a given geography 5 , 6 , and how they depend on behavioral, social, age structure, and other factors of a population. It is worth seeking whether and how these factors relate to the expressed epidemic model rate parameters as phenotypes.…”

“…Infection 1 , 2 , transcription and replication 3 , 4 by SARS-COV-2 involve a number of rate limiting interactions with host cells that are likely to be modulated by mutations in cellular as well as viral genes. At the same time, phylogenetic analysis shows geographic specificity 5 , 6 , indicating that geographic regions may show specific exposure to distinctive SNP combinations, or viral haplotypes, in SARS-COV-2. This specificity suggests a benefit to exploring relationships between duration of the prodromal phase, proportions of asymptomatic cases 7 , 8 , proportions of severe cases, rates of recovery, among other infection attributes 9 , that define temporal progression of compartmental epidemic models, starting with SIR (susceptible–infected–recovered) models 10 .…”