EXOPROT

The conserved 3’-5’ exoribonuclease EXOSC10 (Rrp6 in yeast) is essential for early embryogenesis, development of the forebrain and red blood cells, gametogenesis and cancer cell division. Among the protein’s best-studied substrates are ribosomal RNAs (rRNAs) and small nucleolar RNAs (snoRNAs) important for ribosome biogenesis, and long non-coding RNAs (lncRNAs) involved in B-cell enhancer activity and double strand break (DSB) repair. Our key goal is to understand how the stability of human 3’-5’ exoribonuclease EXOSC10 is regulated, and if external cues such as physical and chemical stress impair ribosome biogenesis and translation elongation by altering the protein’s cellular concentration, hence activity. We hypothesize that EXOSC10 is targeted by the anaphase promoting complex/cyclosome (APC/C)-proteasome pathway and thereby constitutes a molecular link between external cues and effects on ribosome biogenesis and function. Our model predicts that EXOSC10 alleles with altered stability and/or activity may be at the origin of certain ribosomopathies, which are defined as diseases caused by altered structural RNA/protein components of ribosomes or gene products required for ribosome biogenesis. Given that the anti-cancer drug 5-fluorouracil (5-FU) reduces the enzymatic activity of EXOSC10 via substrate inhibition, mutant alleles showing increased stability in cancer cells may also contribute to cancer cell survival and ultimately drug resistance via a compensatory mechanism. Our key objectives are to (1) demonstrate that yeast Rrp6 and mammalian EXOSC10 are extensively regulated at the post-translational level via environmental cues, (2) test our hypothesis that the APC/C-proteasome pathway plays a critical and evolutionarily conserved role in controlling EXOSC10/Rrp6 stability, (3) determine if abnormally stable EXOSC10 alleles alter a cancer cell’s ability to progress through the mitotic cell cycle and to respond to drug treatment, and (4) identify amino acids within the predicted destruction boxes and any of the modified residues in EXOSC10 have been mutated in cancer patients, and study the effect of these alterations on cell division and drug resistance in cultured cells. The project will lead to insight into the regulatory mechanisms that govern EXOSC10 stability and thereby help better understand how nutritional, physical and chemical stress influences ribosome biogenesis and protein translation. The outcome of the proposed work is thus not only of cell- and developmental biological interest but will likely also help improve cancer prognosis and drug treatment and efforts to elucidate molecular mechanisms underlying ribosomopathies in molecular oncology and the field of human developmental disorders.