The primary goal of my lab is to define the basis by which non-coding elements in messenger RNA sequences define differential regulation of gene expression. The model system is early embryogenesis of the nematode Caenorhabiditis elegans. The experimental strategy is to determine the nucleotide binding specificity and assembly mechanism of each protein involved in recognition of the non- coding elements using quantitative in vitro methods. Then, the mRNAs that associate with each protein are independently identified using crosslinked immunprecipitation and/or RNA-immunoprecipitation and array. The functional relevance of the binding specificity is tested in live animals using transgenic reporters that assay for regulation. This approach is the logical opposite of standard forward genetics, yet it enables a quantitative understanding of mRNA discrimination that is not possible using solely in vivo methods. The long term goal of my lab is to delineate the complete wiring diagram of RNA regulatory circuitry in the embryo, and elucidate the regulatory mechanisms that control maternal mRNA translation, localization, and turnover. A necessary first step toward this goal is to identify the RNA targets of each regulatory protein, and determine how they work together to select specific mRNAs for regulation. In this proposal, we focus on the RNA-binding proteins that pattern Notch/glp-1 expression in the embryo (MEX-3, MEX-5, POS-1, SPN-4, and GLD-1). In preliminary work, we have made a several important discoveries relevant to mRNA recognition by these factors that argue cooperative and antagonistic interactions drive recognition of glp-1 transcripts. These results lead to our current hypothesis: Occupancy of the RNA- binding proteins on the glp-1 3'-UTR defines its spatial and temporal expression pattern.
The specific aims outlined in this proposal will test this model, and identify novel regulatory targets of each protein that may contribute to the pleiotropy and disparity of the mutant phenotypes for each of these proteins. Our work will describe basic mechanisms that contribute to the totipotency of embryonic cells, which has relevance to several modern therapeutic strategies. All of the proteins that we propose to study have homologs in mammals, many of which play roles in human development, including placental differentiation, formation of the central nervous system, vascularization, and immunity. Lessons learned from this project may aid in understanding human biology that contributes to inflammatory disease, neurological and psychiatric disorders, and congenital developmental abnormalities. Project Narrative: This proposal describes experiments aimed at understanding the process by which a fertilized egg transforms into a multicellular animal. By defining the regulatory processes that govern initial development, it may be possible to develop new strategies to combat infertility and novel contraceptive methods. Lastly, there is a surprising correlation between RNA regulation during embryogenesis, inflammation response, and myelination in the central nervous system. This work may lead to new breakthroughs relevant to polyinflammatory arthritides including rheumatoid arthritis and other autoimmune disorders including multiple sclerosis.
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