Poster abstracts
Poster number 40 submitted by Paul Russell
Deciphering mechanisms of ribosome turnover during global inhibition of translation elongation in mammalian cells
Paul J. Russell (Ohio State Biochemistry Program, The Ohio State University), Michael G. Kearse (Cellular, Molecular, and Biochemical Sciences Program, The Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology, Center for RNA Biology)
Abstract:
Eukaryotes use several distinct quality control pathways to resolve aberrant ribosomes and mRNAs. For example, the no-go decay mRNA pathway is stimulated after ribosome collisions caused by 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) affects ribosome turnover. Despite steady levels of cell viability 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 50% reduction of ribosomes. 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 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. Additionally, we show using inhibitors that target different stages of translation that this proteasome-dependent decay pathway is stimulated by stalled ribosomes and not primarily by low levels of translation. Moving forward, we will test the hypothesis that globally stalled ribosomes in mammalian cells are ubiquitinated and subsequently degraded by the proteasome and RNA exosome machinery.
Keywords: ribosome turnover, proteasome, quality control