Deconvoluting disparate roles of DDX6 in translational control - PROJECT SUMMARY The putative oncogene DDX6 (RCK, p54) has critical roles in the nervous system, embryonic cells, and somatic cells. The importance of DDX6 is further emphasized by rare pathogenic missense mutations in DDX6 that are associated with developmental delays and intellectual disability in children. DDX6 is a member of the DEAD-box RNA helicase family, which are broadly involved in regulation of messenger RNA (mRNA) and translation. Despite its establishment as critical regulator of translation initiation, the molecular mechanisms that underlie DDX6 function remain unclear. Major roadblocks to this include that translation initiation is a heterogenous, multistep process that requires a dozen components and occurs within a minute. Further, the identity of interacting partners in a given DDX6 regulatory complex determines whether DDX6 promotes or inhibits protein synthesis. This complicates cellular study of DDX6, as DDX6 likely forms multiple distinct regulatory complexes on different mRNAs at any given time. To overcome these roadblocks, I will combine biochemical, single-molecule, and biophysical methods to examine my central hypothesis that DDX6 regulates the initial, rate-limiting step of translation initiation and global mRNA conformation in concert with its interaction partners. In Aim 1, I will identify the step of translation initiation that is regulated by DDX6 and its key partner proteins by using in vitro single molecule fluorescence microscopy and a reconstituted human translation initiation system. In Aim 2, I will examine how DDX6 assembles with partner proteins into distinct regulatory complexes and how this impacts mRNA dynamics. In each Aim, I will determine how pathogenic missense mutations in DDX6 disrupt its function. My proposed experiments will uniquely leverage my background in protein biochemistry and enzymology, the Lapointe lab’s expertise in human translation initiation and single molecule fluorescence microscopy, and the Stoddard lab’s expertise in structural biology and enzymology. My collective findings should elucidate which step of translation initiation is controlled by DDX6, how DDX6 assembles into translation inhibiting and translation stimulating complexes, how distinct DDX6 complex assemblies remodel mRNA, and how missense mutations alter DDX6 function. In parallel, my individualized training plan will provide me with opportunities to expand my research skillset into single molecule techniques and human translation initiation, to grow as a leader and mentor within the Lapointe lab, and prepare me for a career as an independent investigator.
