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水平方面起着重要作用。因此，改变的翻译控制是许多人类疾病的关键因素，包括癌症和神经系统疾病。与基因表达的许多步骤一样，调节翻译的特征是rna结合蛋白（rbp）与特定mrna的特定区域相互作用。在翻译过程中，这些mRNA元件和结合因子究竟如何共同作用影响mRNA的命运，目前尚不清楚。对于多细胞生物中的特化细胞来说尤其如此，因为在体内研究特定细胞的翻译控制是具有挑战性的。我们研究的一个关键目标将是阐明非规范翻译对多细胞生物中特定细胞类型的特化功能的贡献。我们的项目重点将是rna结合蛋白和“非规范翻译因子”eIF2D（也称为Ligatin）。在我们的工作之前，对这类蛋白在多细胞生物中的体内功能或分子靶点几乎一无所知。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复合体选择性地促进上游开放阅读框架（Open Reading Frames, uorf）下游的翻译再启动，从而促进组织生长。在这里，我们基于这些发现和相关的实验方法来研究DENR-MCT-1同源物eIF2D。为了首次在动物身上分析eIF2D的体内功能，我们培育了eIF2DKO果蝇，发现它们是有活力和可生育的，没有明显的发育和形态缺陷。然而，eIF2DKO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明显的缺陷。在eIF2DKO背景下，在运动神经元或肌肉细胞中过表达eIF2D转基因基因可以完全挽救运动缺陷。因此，eIF2D在肌肉和运动神经元中发挥关键作用，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eIF2D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，确定eIF2D在体内运动神经元和肌肉中翻译控制的直接mRNA靶点。随后，我们将使用报告基因分析分析这些mrna的作用机制。最后，我们将结合体内方法来研究eIF2D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。我们希望我们的结果能够为多细胞生物中翻译控制如何支持细胞特异性功能提供重要的见解。此外，我们将建立新的，普遍适用的方法来分析体内特定细胞类型的多细胞生物的翻译控制。

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.