OTUD6 deubiquitinates the 40S ribosomal protein RPS7 to increase protein translation and resistance to cellular stress

Ribosome ubiquitylation is a highly regulated process that is essential for maintaining proper protein translation. While much is known about the role of ubiquitin ligases in these pathways, less is known about the function of deubiquitinases. OTUD6 is an ovarian tumor (OTU) family deubiquitinase that is conserved from yeast (OTU2) to humans (OTUD6A & B). To study OTUD6 in Drosophila, CRISPR/Cas9 was used to create a null mutant, two mutants with abolished catalytic activity, and epitope-tagged wild-type and catalytically dead (DUB-dead) endogenous OTUD6 fly lines. DUB-dead and loss-of-function OTUD6 mutants are markedly sensitive to oxidizing (paraquat) and alkylating (mms) agents that impact protein translation and ribosome function. Co-immunoprecipitation coupled with mass spectrometry in OTUD6.DUB-dead brains, where OTUD6 is highly expressed, identified 40S ribosome and RNA exosome proteins. The most significantly enriched 40S proteins, RACK1 and RPS3, play key roles in ribosome quality control and turnover under cellular stress. Mutation of RACK1 rescued the mms sensitivity of OTUD6 mutants, indicating that OTUD6 is likely a negative regulator of RACK1. RACK1 regulates ubiquitylation of several 40S ribosome subunits: a genetic screen of E3 ligases that ubiquitylate the 40S ribosome in a RACK1 dependent manner revealed that OTUD6.DUB-dead interacts epistatically with three E3 ligases. Two of the E3 ligases, RNF10 and RNF123, are implicated in RPS3 ubiquitylation in ribosome quality control. The additional E3 ligase, NOT4, is implicated in RPS7 ubiquitination. In support of OTUD6 regulating ribosome quality control or turnover, OTUD6 mutants have decreased protein translation, delayed development, and dramatically extended lifespan. Thus, OTUD6 deubiquitinates the ribosomal subunit RPS7, to regulate protein translation and the response to alkylation damage. Additionally, I show for the first time that RACK1 and RNF10 can regulate RPS7 ubiquitination