Regulation of Gene Expression by microRNAs in C. elegans

描述(申请人提供)：在动物发育和疾病中，由一大类小的(~22nt)非编码的microRNAs控制基因表达已经成为一种新的转录后基因调控模式。MicroRNA诱导的沉默复合体(MiRISC)通过部分碱基配对将microRNAs引导到靶mRNAs的3&1非编码区，导致翻译抑制和/或靶标降解。然而，靶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活跃的研究领域。在这项提案中，我们将确定一种新的蛋白质复合体的功能，该复合体结合线虫中的microRNAs及其靶标。该复合体包括miRISC的保守成员AIN-1以及三个高度保守的带有冷休克结构域(CSD)的RNA结合蛋白。我们假设CSD蛋白是抑制microRNA靶标表达的关键因素。为了阐明CSD蛋白的功能，我们将重点研究线虫身体肌肉中miR-1mRNA靶标的调控。我们之前在miR-1及其对神经肌肉接头突触信号的调节方面的工作为我们目前的提议建立了实验框架。我们将测试在miR-1靶向表达、与AIN-1复合体结合以及募集到P小体中对CSD蛋白的需求。我们将在以下两个目标中验证我们的假设：在目标1中，我们将研究CSD蛋白在调节miR-1靶标表达和与AIN-1复合体结合方面的功能。我们假设CSD蛋白直接或间接地将miR-1靶向mRNAs招募到AIN-1复合体，用于随后的靶向抑制。在目标2中，我们将测试CSD蛋白在AIN-1复合体形成和募集到P小体中的作用，P小体是抑制microRNA靶向mRNAs的细胞质中心。我们假设，CSD蛋白与mRNA底物的结合是AIN-1复合体形成和靶向P小体的必要步骤。最后，我们将检验这一假设，即单个CSD蛋白是否形成不同的、模块化的AIN-1复合体，也许是作为调节microRNA靶标的一种方式。我们期望这些研究将为如何识别和指定翻译抑制或降解的microRNA靶标的保守机制提供见解。与公共卫生相关：我们发现了一种新的蛋白质复合体，我们假设它具有调节microRNAs基因靶标的功能。MicroRNAs代表了一个由所有动物基因组编码的新的小RNA分子超家族。通过与它们的目标mRNAs结合，microRNAs阻止目标基因被翻译成蛋白质。这种新的基因调控模式影响了动物发育中的多种生物学过程，并与包括癌症和心脏功能障碍在内的越来越多的疾病状态有关。然而，执行microRNA介导的靶基因调控所需的机制和细胞机制仍未完全确定。我们建议研究我们新的AIN-1蛋白复合体及其在我们的模式生物秀丽线虫体内肌肉的microRNA靶标结合和调节中的关键作用。

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. PUBLIC HEALTH RELEVANCE: We have discovered a novel protein complex that we hypothesize functions to regulate the gene targets of microRNAs. MicroRNAs represent a new superfamily of small RNA molecules encoded by all animal genomes. By binding to their target mRNAs, microRNAs prevent the target genes from being translated into proteins. This new paradigm of gene regulation affects a multitude of biological processes in animal development and has been implicated in an increasing number of disease states including cancer and cardiac dysfunction. However, the mechanisms and the cellular machinery required to perform microRNA-mediated target gene regulation remain to be fully characterized. We propose to study our novel AIN-1 protein complex and its critical roles in microRNA target binding and regulation in the body muscles of our model organism, C. elegans.