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

项目总结/文摘:

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