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Abstract

Ribonucleoprotein (RNP) granules are membraneless compartments that form inside the cyto- or nucleoplasm by the process of phase separation. RNP granules play essential roles in cell organization and physiology and their misregulation can have detrimental effects including protein misfolding and aggregation. Even though our knowledge about RNP granules has increased greatly in the past years there are still many open questions about the mechanisms involved in their formation, dissolution, and properties. There are indications that RNA helicases could play a crucial role in regulating the properties of RNP granules. Many RNA helicases can be found in different types of RNP granules. Furthermore, they have the ability to bind and remodel secondary structures of RNA, one of the main components of RNP granules. This gives them the potential ability to influence RNP granule formation, dissolution, and properties. In my PhD thesis, I analysed the human DEAD-box helicase DDX3X, which is known to be present in stress-inducible RNP granules. For this, I established a purification method for DDX3X-WT as well as two cancer related-variants of DDX3X. By biochemical analysis, I could demonstrate that the cancer-related variants are partially or fully defective in their enzymatic activity. I could show that DDX3X forms condensates at physiologically relevant conditions in vitro and that the phase separation propensity is independent on the enzymatic activity. However, analysis of the material properties of condensates formed by DDX3X-WT and the two cancer-related variants in different conditions revealed significant differences. This indicates that the enzymatic activity of DDX3X is relevant for the material properties of these condensates. Furthermore, I set up an in vitro system to mimic stress-inducible RNP granules, using G3BP1 and RNA. This assay revealed that condensates formed by G3BP1 in the presence of heat-aggregated RNA exhibit solid-like features. I could show that DDX3X-WT, but not the cancer-related variants, collaborates with G3BP1 and promotes a solid to liquid phase transition of these solid-like condensates. Taken together, this suggests that the RNA helicase DDX3X can regulate the material properties of RNP granules using its enzymatic activity. This reveals a potential mechanism how DEAD-box helicases could regulate RNP granules formation, dissolution and their material properties.