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

基因表达的翻译控制在决定细胞蛋白质水平中起主要作用。因此，改变的翻译控制是许多人类疾病，包括癌症和神经障碍的关键因素。与基因表达的许多步骤一样，受调控的翻译以与特定mRNA的特定区域相互作用的RNA结合蛋白（RBP）为特征。这些mRNA元件和结合因子如何在翻译过程中共同作用以影响mRNA的命运仍然知之甚少。这对于多细胞生物体中的特化细胞尤其如此，因为研究体内特定细胞内的翻译控制是具有挑战性的。 我们研究的一个关键目标是阐明非经典翻译对多细胞生物中特定细胞类型的专门功能的贡献。我们项目的重点将是RNA结合蛋白和“非经典翻译因子”eIF 2D（也称为Ligatin）。在我们的工作之前，几乎没有人知道这种蛋白质在多细胞生物体中的体内功能或分子靶点。我们最近在果蝇中使用了一种体内/体外联合方法，表明DENR-MCT-1复合物选择性地促进上游开放阅读框架（uORF）下游的翻译重新起始，以促进组织生长。在此，我们建立了这些发现和相关的实验方法来研究DENR-MCT-1同源物，eIF 2D。为了分析eIF 2D在动物体内的功能，我们第一次产生了eIF 2DKO果蝇，并发现它们是可行的和可育的，没有明显的发育或形态缺陷。然而，eIF 2DKO幼虫在运动行为和运动神经元与肌肉之间的突触通讯方面表现出显著的缺陷。运动缺陷可以通过在eIF 2DKO背景下在运动神经元或肌肉细胞中过表达eIF 2D转基因来完全挽救。因此，eIF 2D在肌肉和运动神经元中发挥关键作用，以促进驱动正常运动行为的突触信号传导。我们现在的中心目标是将这些高度特异性的体内表型与运动神经元和肌肉中eIF 2D对特定mRNA的翻译控制联系起来。为了实现这一目标，我们将首先进行实验，以确定eIF 2D的直接mRNA目标的翻译控制在运动神经元和肌肉在体内。随后，我们将使用报告分析来剖析这些mRNA的功能机制。最后，我们将使用体内方法的组合来研究eIF 2D对特定mRNA的调节如何与其在运动神经元和肌肉中的功能相关。我们希望我们的研究结果提供了重要的洞察翻译控制如何支持多细胞生物体中的细胞特异性功能。此外，我们将建立新的，普遍适用的方法来分析体内多细胞生物的特定细胞类型的翻译控制。

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.