Understanding the molecular interactions and mechanisms of the RNA helicase DDX6 in posttranscriptional regulation

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RNA fate determination is at the center of gene expression regulation, influencing the stability, localization, and translation efficiency of mRNAs. Here, we propose to characterize the RNA helicase DDX6 and its role in posttranscriptional regulation. DDX6 is known to be involved in mRNA localization and storage in different protein-mRNA granules, recruitment of mRNA degradation enzymes, and translation repression through the miRNA pathway. Despite its importance the RNA-target specificity and the detailed molecular functions of DDX6 remain elusive. We will deliver three major milestones that are independent from each other, but feed into a system-wide description of DXX6s specificity and regulation at the example of human HEK293 cells. We will map the mRNA interactome of DDX6 and mutant variants using protein-RNA crosslinking, immunoprecipitation, and next-generation sequencing to identify mRNAs where DDX6 binds, the region within the mRNA, and how the repertoire of putative targets changes. We will characterize the repertoire of protein interaction partners and DDX6-protein complexes through proximity biotinylation followed by mass spectrometry. To characterize the global molecular impact of DDX6 function, we will deplete the helicase and determine changes in mRNA half-lives and ribosome occupancy. These three assays provide an overview of the changes that putative target transcripts may undergo with respect to their mRNA stability and translation. None of these proposed studies has been performed before in a comprehensive, system-wide manner, and their combination will provide synergistic information on changes in the functions and molecular targets of DDX6, depending on its interactions with other putative regulators. As RNA-binding proteins are still systematically understudied, we will provide important insights of the roles of a key player in post-transcriptional regulation of gene expression.