Regulation by post-translation modifications in response to stress

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Project Summary/Abstract Ubiquitin ligases (E3s) represent a diverse and conserved group of enzymes, with over 600 members. These ligases collectively attach the small protein ubiquitin to more than twenty five percent of the proteome, thereby regulating the stability or activity of each target. Despite the importance of this set of enzymes, only a small percentage of ubiquitin ligases have well-characterized biological functions. We have conducted a CRISPR screen examining the sensitivity of mutations of genes encoding human ubiquitin ligases and deubiquitinating enzymes to a panel of inhibitors covering a broad range of biological pathways. From this screen, we identified a CUL5 specificity subunit, called WSB2, whose mutation renders cells sensitive to inhibitors of nuclear export and mitochondrial function. Preliminary studies identified two sets of proteins whose levels are affected by WSB2: the apoptotic factor BIM and the mRNA export proteins SNIP1 and THRAP3 (two members of the SNARP complex). In this application, we will determine whether these are independent substrates of the CUL5-WSB2 complex, or alternatively, whether targeting of SNIP1/THRAP3 affects BIM mRNA levels or localization. If SNIP1 and/or THRAP3 are direct substrates, we will examine the regulation of this ubiquitination event.