Regulation of Gene Expression by microRNAs in C. elegans

描述（由申请人提供）：由大类小（~ 22 nt）非编码microRNA控制基因表达已成为动物发育和疾病中转录后基因调控的新模式。microRNA诱导的沉默复合物（miRISC）通过部分碱基配对将microRNA引导至靶mRNA的3< UTR，并导致翻译抑制和/或靶降解。然而，目标识别和结合的步骤，以及miRISC和其他RNA相关因子的作用仍然是研究的活跃领域。在这个提议中，我们将确定一种新的蛋白质复合物的功能，这种蛋白质复合物可以结合线虫C.优雅该复合物包括AIN-1，miRISC的保守成员，以及三个具有冷休克结构域（CSD）的高度保守的RNA结合蛋白。我们假设CSD蛋白是抑制microRNA靶点表达的关键因子。为了阐明CSD蛋白的功能，我们将重点研究miR-1 mRNA靶点在C.优雅我们先前关于miR-1及其在神经肌肉接头处的突触信号调节的工作为我们当前的提议建立了实验框架。我们将测试CSD蛋白在miR-1靶向表达、与AIN-1复合物的结合以及向P体的募集中的需求。我们将在以下两个目标中测试我们的假设：在目标1中，我们将研究CSD蛋白在调节miR- 1靶向表达和与AIN-1复合物结合中的功能。我们假设CSD蛋白直接或间接地将miR-1靶mRNA募集到AIN-1复合物中，用于随后的靶抑制。在目标2中，我们将测试CSD蛋白在AIN-1复合物形成和募集到P体中的作用，P体是抑制microRNA靶mRNA的细胞质中心。我们假设CSD蛋白与mRNA底物的结合是AIN-1复合物形成和靶向P体的必要步骤。最后，我们将测试单个CSD蛋白是否形成不同的模块化AIN-1复合物的假设，这可能是调节microRNA靶标子集的一种方式。我们期望这些研究将提供对microRNA靶标如何被识别和指定用于翻译抑制或降解的保守机制的见解。

DESCRIPTION (provided by applicant): The control of gene expression by the large class of small (~22nt), noncoding microRNAs has emerged as a new mode of post-transcriptional gene regulation in animal development and disease. The microRNA-induced silencing complex (miRISC) guides microRNAs to the 3< UTR of target mRNAs through partial base pairing and leads to translational inhibition and/or target degradation. However, the steps of target recognition and binding, and the role of miRISC and other RNA-associated factors remain active areas of investigation. In this proposal we will determine the function of a novel protein complex that binds microRNAs and their targets in the nematode, C. elegans. This complex includes AIN-1, a conserved member of miRISC, as well as three highly conserved RNA binding proteins with Cold Shock Domains (CSD). We hypothesize that the CSD proteins function as key factors for repressing the expression of microRNA targets. To elucidate the function of the CSD proteins, we will focus our studies on the regulation of miR-1 mRNA targets in the body muscle of C. elegans. Our previous work on miR-1 and its regulation of synaptic signaling at the neuromuscular junction establishes the experiment framework for our current proposal. We will test the requirement for the CSD proteins in miR-1 target expression, binding to the AIN-1 complex, and recruitment to P bodies. We will test our hypothesis in the following two aims: In Aim 1, we will investigate the function of the CSD proteins in the regulation of miR- 1 target expression and binding to the AIN-1 complex. We hypothesize that the CSD proteins recruit the miR-1 target mRNAs, directly or indirectly, to the AIN-1 complex for subsequent target repression. In Aim 2, we will test the role of the CSD proteins in AIN-1 complex formation and recruitment to the P bodies, the cytoplasmic centers for repression of microRNA target mRNAs. We hypothesize that binding of mRNA substrates by the CSD proteins is a required step for AIN-1 complex formation and targeting to P bodies. Finally, we will test the hypothesis of whether individual CSD proteins form distinct, modular AIN-1 complexes, perhaps as a way to regulate subsets of microRNA targets. We expect that these studies will provide insights into the conserved mechanisms of how microRNA targets are recognized and designated for translational inhibition or degradation.