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Specific catalytically impaired DDX3X mutants form sexually dimorphic hollow condensates
Michael C Owens1,2, Hui Shen1,3, Amber Yanas1,2
1Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Abstract:
Mutations in the RNA helicase DDX3X, implicated in various cancers and neurodevelopmental disorders, often impair RNA unwinding and translation. However, the mechanisms underlying the impairment and the differential interactions of DDX3X mutants with wild-type (WT) X-linked DDX3X and Y-linked homolog DDX3Y remain elusive. This study reveals that specific DDX3X mutants more frequently found in disease form distinct hollow condensates in cells. Using a combined structural, biochemical, and single-molecule microscopy study, we show that reduced ATPase and RNA release activities contribute to condensate formation and these catalytic deficits result from inhibiting the catalytic cycle at multiple steps. Proteomic investigations further demonstrate that these hollow condensates sequester WT DDX3X/DDX3Y and other proteins crucial for diverse signaling pathways. WT DDX3X enhances the dynamics of heterogeneous mutant/WT hollow condensates more effectively than DDX3Y. These findings offer valuable insights into the catalytic defects of specific DDX3X mutants and their differential interactions with wild-type DDX3X and DDX3Y, potentially explaining sex biases in disease.
Insights
Mutant RNA helicase DDX3X forms abnormal condensates, impairing its function in cancer and developmental disorders. Wild-type DDX3X interacts differently with these mutants than DDX3Y, potentially explaining disease sex biases.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Mutations in RNA helicase DDX3X are linked to cancers and neurodevelopmental disorders.
- The precise mechanisms of DDX3X dysfunction and interactions with its homologs (DDX3X and DDX3Y) are not fully understood.
Purpose of the Study:
- To investigate the impact of disease-associated DDX3X mutations on its structure, function, and interactions.
- To elucidate the role of DDX3X mutants in condensate formation and protein sequestration.
- To explore the differential effects of wild-type DDX3X and DDX3Y on mutant DDX3X behavior.
Main Methods:
- Structural, biochemical, and single-molecule microscopy techniques were employed.
- Proteomic analysis was used to identify sequestered proteins.
- Cellular assays were performed to study condensate formation and dynamics.
Main Results:
- Specific DDX3X mutants form distinct hollow condensates, associated with reduced ATPase and RNA release activities.
- These condensates inhibit the DDX3X catalytic cycle at multiple steps.
- Hollow condensates sequester wild-type DDX3X/DDX3Y and other signaling proteins.
- Wild-type DDX3X more effectively enhances the dynamics of mutant/wild-type condensates compared to DDX3Y.
Conclusions:
- Disease-associated DDX3X mutations lead to catalytic deficits and aberrant condensate formation.
- These condensates sequester essential proteins, disrupting cellular signaling.
- Differential interactions between mutant DDX3X and its wild-type homologs may underlie sex-biased disease prevalence.
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