Medulloblastoma-Associated Mutations In The Dead Box Rna Helicase Ddx3x Impair Protein Translation

Whole genome sequencing technologies recently identified novel recurrent mutations in childhood medulloblastomas (MB), including missense mutations in the DEAD box RNA helicase DDX3X. In the present study, we used complementary genomic approaches to illuminate normal DDX3X function and the impact of cancer-associated mutations. We used CLIPseq to identify the RNA targets of endogenous DDX3X. This analysis revealed that DDX3X primarily binds mature mRNAs and, further, that the distribution of DDX3X binding sites in targets spans the full length of these mRNAs, with enrichment in the coding regions, suggesting a role for DDX3X in translation elongation. Consistent with this observation, we detected a fraction of wild type DDX3X in polysomes by sedimentation experiments. In contrast, MB-associated DDX3X mutants appear to associate poorly with polysomes, but induce spontaneous stress granule (SG) formation in cells, suggesting a role of mutant DDX3X in triggering translation silencing. In fact, the RNA binding capabilities of DDX3X are still preserved in the context of cancer-associated mutations further supporting a role of mutant DDX3X in repressing translation of specific RNAs by retaining them within SGs. Interestingly, gene ontology analyses find mRNAs encoding translation factors themselves to be highly enriched among DDX3X RNA targets, suggesting that perturbations of DDX3X functions could have far-reaching impact on cellular translation. Indeed, knocking down DDX3X with siRNA or overexpressing a mutant led to a significant impairment in global translation as measured by S35-methionine metabolic labeling. Furthermore, assessment of whole-genome protein translation by ribosomal profiling analyses in cells overexpressing cancer-associated mutant DDX3X confirmed a decreased translation rate of direct DDX3X mRNA targets in particular, but also numerous other mRNAs. Finally, we have generated an allelic series of transgenic fruit flies expressing wild type or mutant versions of human DDX3X or its Drosophila ortholog Belle, and have captured mutation-dependent phenotypes when these transgenes are expressed in specific tissues. Together, our data show that DDX3X regulates the translation of a key subset of mRNAs and thereby indirectly influences cellular translation globally, and that this function is perturbed by cancer-associated mutations. This abstract is also presented as poster C48. Citation Format: Yasmine A. Valentin-Vega, Matthew Parker, Michael Rusch, Demelza Smeeth, Nam Chul Kim, Jinghui Zhang, Richard J. Gilbertson, Joseph P. Taylor. Medulloblastoma-associated mutations in the DEAD box RNA helicase DDX3X impair protein translation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR11.