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

项目摘要/摘要： 尽管生殖技术在过去几十年里取得了惊人的进步，但大约 美国仍有10%的育龄夫妇不育。不幸的是，现有的受助者 生殖技术，如体外受精，并不是解决这一重大社会问题的灵丹妙药 有问题。一些严重的差距仍然存在于理解分子机制的细节上 生殖过程，这是认识不孕不育原因的一个重大障碍。因此，我们 需要极大地提高我们的生殖基础知识，从如何受精开始 配子的产生与健康胚胎发生的早期阶段的方法有关。因此， 这项研究的长期目标是更全面地了解 以线虫为模型系统的配子发生。线虫是一种非常适合解决的遗传系统 这些特殊的研究问题是因为透明的动物允许实时、活体观察 卵子发生、受精和胚胎发生。我们最近发现了一种保守的RNA结合蛋白(RBP) 在线虫中，ETR-1似乎在生殖和生育中发挥重要作用，但其 生殖功能此前仍未确定。ETR-1的人类同源物CUGBP1是 与强直性肌营养不良症有关，这是一种肌肉退行性疾病，各种男性和 据报道，女性患者也有生育问题。ETR-1在线虫中大量表达 由于限制性商业惯例是基因表达的重要调节者，因此我们假设ETR-1是一种 正常基因调控的基本组成部分，发生在卵子发生过程中，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，靶基因调控失调(S)导致生殖异常。为了解决这个问题，我们 制定了两个具体目标。具体目标1将集中在确定ETR-1在性腺中的作用 发展和功能。具体目标2将侧重于确定ETR-1在 通过确定ETR-1的体内RNA靶点来实现配子发生。在目标1中，详细表型 性腺的特征将在ETR-1耗尽后通过RNAi以及系统地进行 ETR-1基因部分缺失。利用CRISPR/Cas-9技术内源性GFP-Tag特异性 在对ETR-1剪接异构体进行实时成像分析后，我们将确定体内对 蛋白。ETR-1的作用机制将在目标2中通过识别RNA靶标来进一步探索 并阐明了优先目标的作用/功能。这方面的另一个重要长期目标是 建议培养本科生对发育生物学研究领域的兴趣。这个 该提案将促进未被充分代表的少数族裔学生在其职业生涯的早期大量参与 在霍华德大学，并激励他们加入研究努力。这项研究具有很强的针对性

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