mRNA assembly in Drosophila germ granules

mRNA ASSEMBLY IN DROSOPHILA GERM GRANULES Development of every species relies critically on spatially organized messenger RNAs (mRNAs). Indeed, even a single asymmetrically distributed mRNA can specify the morphology of cells, the body plan of a developing organism and cell lineages across metazoans. RNA binding proteins (RBPs) typically organize mRNAs and recruit them to subcellular structures. However, mRNAs can also self-organize into multi-mRNA assemblies independently of other cellular components. mRNA assemblies are found in healthy cells in different species and are often associated with RNA granules, membraneless ribonucleoprotein (RNP) particles that regulate translation and stability of mRNAs. mRNA assemblies are also a hallmark of pathogenesis in human repeat expansion disorders such as myotonic dystrophy 1 (MD1), amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia (SCA). These findings indicate that mRNA assembly is an inherent property of an mRNA that could be biologically relevant. However, the exact mechanism and potential biological functions of mRNA assembly is unclear. mRNAs have been shown to self-assemble by phase separation, a process akin to oil-and- water de-mixing. Here, RNA:RNA interactions, which promote intermolecular (trans) base-pairing and secondary (cis) RNA structures have been implicated in driving mRNA assembly. In addition, in vitro data suggest that RNA helicases, which are core constituents of RNA granules are thought to dissolve RNA:RNA interactions in granules. In light of these findings, several critical questions arise. How do mRNAs self-assemble in granules designed to prevent RNA:RNA interactions? What is the role of mRNA assemblies in RNA granules? What is the function of trans and cis RNA:RNA interactions and RNA helicases in the formation and function of mRNA assemblies and RNA granules? The five-year goal of this study is to answer these questions and determine the mechanism and biological function of mRNA assembly in Drosophila germ granules. mRNA assemblies tend to form as small clusters that are acutely sensitive to the concentration and the environment in which they form. Thus, the central challenge in studying mRNA assembly is that methods must be employed that enable examination of this process in vivo and with high resolution and sensitivity. We will use genetic and quantitative, super-resolution imaging approaches to analyze mRNA assembly in intact cells and within their natural cellular context. We aim to uncover new insight into how organisms harness mRNA assemblies to promote development and how misregulation of mRNA assembly could contribute to human diseases, such as MD1, ALS and SCA.