Identifying the cellular machinery responsible for ribosome turnover during global inhibition of translation elongation in mammalian cells
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Date
2024-05
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The Ohio State University
Abstract
Eukaryotes use several distinct quality control pathways to resolve aberrant ribosomes and mRNAs. For example, the no-go decay mRNA pathway is stimulated during ribosome collisions caused by slowed/stalled ribosomes translating damaged or truncated mRNAs. Separate decay pathways for non-functional 40S and 60S subunits containing rRNA mutations affecting decoding and peptidyl transferase activity, respectively, have also been elucidated. To our knowledge, whether eukaryotes have also evolved a quality control pathway to sense and process globally stalled ribosomes is unclear; however, such a pathway would be advantageous to eukaryotes during exposure to natural elongation inhibitors such as ricin and diphtheria toxin. Here, we test how prolonged global inhibition of elongation using cycloheximide (CHX), which binds to the E site within the 60S subunit and blocks eEF2-mediated translocation of the 80S ribosome, affects ribosome turnover. Despite steady levels of cell viability and reducing potential and that mammalian ribosomes have been classically characterized of having a half-life of 3-5 days, a single 24 hr high dose of CHX resulted in a stark loss of ribosomes. Polysome analysis of HeLa cells treated with CHX for 24 hrs showed a 50% reduction in 40S subunits, 60S subunits, 80S monosomes, and polysomes. Depletion of ribosomes from the same treatment in A549 cells was also evident when assessing rRNA levels by denaturing agarose gel electrophoresis and ribosomal proteins (from both the 40S and 60S subunits) by Western blot. Literature supports that ribosomes can be degraded by autophagy/ribophagy and the proteasome. Upon testing various inhibitors of both pathways, only proteasome inhibitors rescued rRNA levels. These data suggest that both ribosomal subunits of inhibited 80S ribosomes are degraded by the proteasome upon prolonged CHX treatment. We hypothesize that globally stalled ribosomes in mammalian cells are ubiquitinated and subsequently degraded by the proteasome and RNA exosome machinery. In my honors research, I tested this hypothesis by completing a small targeted siRNA screen using two independent siRNAs in A549 cells against known ubiquitin E3 ligases shown in the literature to be involved in ribosome quality control (i.e., CUL3, CUL4A, RNF10, UBE2O, ZNF598) to identify the ubiquitin E3 ligase responsible for driving ribosome turnover. I also tested the role of the major eukaryotic exoribonucleases (i.e., XRN1, DIS3, DIS3L, DIS3L2) by siRNA knockdown to identify contribute to ribosome turnover. A non-targeting (scrambled) siRNA was used as a negative control. Knockdown efficiency was determined by measuring mRNA levels by RT-qPCR. Of the ubiquitin E3 ligases tested, only CUL3 was found to partially rescue CHX-mediated ribosome turnover using two independent siRNAs. The screen of the major eukaryotic exoribonucleases that regulate RNA turnover in the cytosol did not reveal any specific factors that contribute to rescue of CHX-mediated ribosome turnover.
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Keywords
Translation, Ribosome, Proteasome, Ubiquitin