Defining the novel role for the RNA binding protein ETR-1 in C elegans gametogenesis

项目总结/摘要： 尽管在过去的几十年里，生殖技术取得了惊人的进步， 在美国，10%的育龄夫妇仍然不育。不幸的是，现有的援助 生殖技术，如体外受精，并不是解决这一重大社会问题的万灵药。 问题.在理解这些分子机制的细节方面仍然存在一些严重的差距， 生殖过程，这是认识不孕症原因的一个重大障碍。所以我们 需要大大提高我们的生殖基础知识，从如何受精能力， 配子产生的方法，其中的早期阶段的健康胚胎发生发生。因此， 这项研究的长期目标是更全面地了解参与的因素， 配子发生的C. elegans作为一个模型系统。C. elegans是一个理想的遗传系统， 这些特殊的研究问题，因为透明的动物允许真实的时间，在体内观察 卵子发生、受精和胚胎发生。我们最近发现了一个高度保守的RNA结合蛋白（RBP） in C. Elegans，ETR-1，似乎在生殖和生育中起着重要作用，但其 生殖功能仍然是以前没有特征的。ETR-1的人类同源物CUGBP 1是 涉及强直性肌营养不良症，一种肌肉变性疾病，其中各种男性和 据报道，女性患者也存在生育问题。ETR-1在C. elegans 由于RBP是基因表达的重要调节因子，因此我们假设ETR-1是一种基因表达的调节因子。 卵子发生过程中正常基因调控的重要组成部分，可能是早期胚胎 发展我们的中心假设是ETR-1在卵子发生过程中是必需的，并且，在缺乏ETR-1的情况下， ETR-1，靶RNA的功能失调导致生殖异常。为了解决这个问题，我们 我们有两个具体的目标。具体目标1将重点确定ETR-1在性腺中的作用， 发展和功能。具体目标2将集中在确定ETR-1功能的机制， 通过确定ETR-1的体内RNA靶点来研究配子发生。在目标1中，详细的表型 性腺的表征将在通过RNAi耗尽ETR-1后进行，以及通过系统性的细胞学方法进行。 etr-1基因的部分缺失。使用CRISPR/Cas-9技术内源性GFP标签特异性 ETR-1剪接异构体，然后进行实时成像分析，我们将确定ETR-1剪接异构体的体内要求。 蛋白ETR-1的作用机制将在Aim 2中通过识别RNA靶点进一步探索 并阐明优先目标的作用/功能。另一个重要的长期目标是 建议旨在培养本科生对发育生物学研究的兴趣。的 该提案将促进代表性不足的少数民族学生在职业生涯早期的大量参与 在霍华德大学，并激励他们加入研究的努力。这项研究具有很强的

PROJECT SUMMARY/ABSTRACT: Despite fantastic advancement made in reproductive technologies within the last few decades, approximately 10% of reproductive age couples in the United States still remain infertile. Unfortunately the existing assisted reproductive technologies, such as in vitro fertilization, are not the cure-all answer to this significant societal problem. Some serious gaps remain in understanding the details of the molecular mechanisms underlying the reproductive process, and this represents a significant hurdle to realizing the causes of infertility. Therefore, we need to greatly enhance our fundamental knowledge of reproduction, from how a fertilization-competent gamete is generated to the methods by which the early stages of healthy embryogenesis occur. As such, the long-term goal of this research is to gain a more comprehensive understanding of the factors involved in gametogenesis using C. elegans as a model system. C. elegans is an ideally suited genetic system to address these particular research questions because the transparent animal allows real time, in vivo observation of oogenesis, fertilization and embryogenesis. We recently identified a well-conserved RNA binding protein (RBP) in C. elegans, ETR-1, which seems to play an essential role in reproduction and fertility, but whose reproductive function remains previously uncharacterized. The human homologue of ETR-1, CUGBP1, is implicated in myotonic muscular dystrophy, a disease of muscle degeneration in which various male and female patients have also been reported to have fertility issues. ETR-1 expresses profusely in the C. elegans gonad, and as RBPs are important regulators of gene expression, we therefore assume that ETR-1 is an essential component of normal gene regulation occurring during oogenesis and, potentially, early embryonic development. Our central hypothesis is that ETR-1 is required during oogenesis, and that, in the absence of ETR-1, dysfunctional regulation of the target RNA(s) results in reproductive abnormalities. To address this, we have devised two specific aims. Specific Aim 1 will focus on determining the role of ETR-1 in gonad development and function. Specific Aim 2 will focus on identifying the mechanism of ETR-1 function in gametogenesis by determining the in vivo RNA targets of ETR-1. In Aim 1, detailed phenotypic characterization of gonads will be performed following depletion ETR-1 via RNAi, as well as by systematic deletion of portions of the etr-1 gene. Using CRISPR/Cas-9 technology to endogenously GFP-tag specific ETR-1 splice isoforms followed by live imaging analysis, we will determine the in vivo requirements of the protein. The mechanism of ETR-1 action will be further explored in Aim 2 through identifying the RNA targets of ETR-1 and elucidating the roles/functions of prioritized targets. Another important long-term goal of this proposal is to foster undergraduate student interest in the area of developmental biology research. The proposal will facilitate substantial participation of underrepresented minority students in their early career years at Howard University and motivate them to join in the research endeavors. This research has strong