Translational control by eIF2D: connecting mRNA targets to cell-specific functions

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Translational control of gene expression plays a major role in determining cellular protein levels. Accordingly, altered translational control is a key element of many human diseases, including cancer and neurological disorders. Like many steps of gene expression, regulated translation features RNA-binding proteins (RBPs) that interact with specific regions of particular mRNAs. How exactly these mRNA elements and bound factors operate together to influence mRNA fate during translation remains poorly understood. This is particularly true for specialized cells in multicellular organisms, since it is challenging to study translational control within specific cells in vivo. A key goal of our study will be to elucidate the contribution of non-canonical translation to specialized functions of specific cell types in a multicellular organism. The focus of our project will be the RNA-binding protein and “non-canonical translation factor” eIF2D (also known as Ligatin). Prior to our work almost nothing was known about in vivo functions or molecular targets of this protein class in multicellular organisms. We recently used a combined in vivo/in vitro approach in Drosophila to show that the DENR-MCT-1 complex selectively promotes translation reinitiation downstream of upstream Open Reading Frames (uORFs) to promote tissue growth. Here, we build on these findings and associated experimental approaches to study the DENR-MCT-1 homolog, eIF2D.To analyze in vivo functions of eIF2D for the first time in animals, we generated eIF2DKO Drosophila, and found that they are viable and fertile with no gross developmental or morphological defects. However, eIF2DKO larvae display striking deficits in locomotion behavior and synaptic communication between motor neurons and muscles. Locomotion defects can be fully rescued by overexpressing an eIF2D transgene in motor neurons or muscle cells in the eIF2DKO background. Thus, eIF2D plays a crucial role in muscles and motor neurons to promote synaptic signaling that drives normal locomotion behavior. Our central objective now will be to connect these highly specific in vivo phenotypes to translational control of specific mRNAs by eIF2D in motor neurons and muscles. To achieve this objective we will first perform experiments to identify eIF2D’s direct mRNA targets for translational control in motor neurons and muscles in vivo. Subsequently, we will dissect the mechanism functioning on these mRNAs using reporter assays. Finally, we will use a combination of in vivo methods to investigate how regulation of specific mRNAs by eIF2D relates to its function in motor neurons and muscles. We expect our results to provide significant insight into how translational control supports cell-specific functions in multicellular organisms. Moreover, we will establish new, generally applicable methods for analyzing translational control in specific cell types of multicellular organisms in vivo.