Role of the ubiquitin system in stress granule turnover

To ensure survival, cells evolved a number of adaptive response mechanisms to cope with various stressful conditions. Upon proteotoxic stress, cells reduce bulk translation and redirect the translation machinery to produce proteins involved in the repair and elimination of damaged proteins. Cytoplasmic membrane-less organelles called stress granules (SGs) are formed in response to various stress conditions, such as heat, osmotic, oxidative stress, and viral infections. SGs contain and protect untranslated messenger ribonucleoprotein (mRNP) complexes, enabling the rapid re-start of translation once the stress ceased. SGs are mainly involved in maintaining cellular homeostasis by protecting mRNP complexes from degradation and enabling rapid re-start of translation once the stress ceased. SG formation and disassembly are tightly regulated by various posttranslational modifications, such as methylation and phosphorylation. In this thesis, the role of the ubiquitin system in SG dynamics was analysed. It was shown that ubiquitin conjugates associate with SGs induced by different stress conditions. Next, the functions of Ub system components in SG turnover were systematically analysed by pharmacological inhibition. It was found that the efficient clearance, but not formation of SGs induced by arsenite or heat stress requires ongoing ubiquitylation and the activity of the ATPase p97, the 26S proteasome, and deubiquitylating enzymes (DUBs). Furthermore, novel DUBs and E3 ligases associating with SGs and regulating SG dynamics were identified. The DUB VCPIP1 was found to be involved in the efficient clearance of SGs induced by different stress conditions, whereas OTUD7B was found to specifically promote the clearance of arsenite-induced SGs. It was shown that the E3 ligase MKRN2, ZNF598, and RNF214 are involved in the ubiquitylation of SG-associated proteins and that the depletion of RNF214 alone or the co-depletion of these E3 ligases in different combinations impaired SG clearance. Taken together, these results demonstrate that ubiquitylation is a key posttranslational modification regulating SG dynamics.