The specific recognition of nucleic acids by proteins drives a variety of cellular processes and is crucial for controlling both transcription of genes, through DNA-binding, and their post- transcriptional regulation, through RNA-binding. While a sequence of nucleic acids is often thought of as a two-dimensional string that is read out by proteins, all nucleic acids have complex three-dimensional structures that are recognized by proteins. The goal of this research project is to determine the role of the sequence and structure of mRNA cis-regulatory elements for recognition by trans-acting factors such as RNA-binding proteins (RBP) to effect post-transcriptional regulation in vivo in C. elegans. Our research project has three aims, with the overarching theme of increasing the scientific community's understanding of post-transcriptional regulation in germ-cell formation and early embryogenesis. First, we will use a new method for determining RBP binding sites with high-resolution, in vivo PAR-CLIP, to find specific in vivo binding targets of RBPs. We will initially characterize FOG-1, which plays a role in spermatogenesis and early germline proliferation in the developing embryo. Second, this high-resolution binding data, in combination with binding data from outside resources, will be used with sequence and structural comparisons of the transcriptome from multiple species to determine how conserved sequence and intrinsic RNA structure act to regulate binding by RBPs. Finally, we will look at binding to the entire transcriptome in the germline to determine which genes that exhibit high-enrichment of being bound by RBPs are targeted for post-transcriptional regulation, and use binding predictions to determine which combination of regulatory factors bind to functionally related transcripts in our high-confidence subset. By combining a new methodology to determine high-resolution binding sites with multiple sources of existing data, we will be able to better understand the ways in which sequence and structure of the germline transcriptome are recognized by RBPs, thereby mediating important post-transcriptional regulation of their localization, translation, and/or stability. PUBLIC HEALTH RELEVANCE: My project aims to understand how the shape and sequence of gene transcripts are involved in recognition by RNA-binding proteins in post-transcriptional regulation of germ-cell formation and early embryogenesis in C. elegans. Cross-species sequence and structure of mRNA cis-regulatory elements, including human sequences, are an integral part of the project. We plan to obtain high-resolution binding data for RNA binding proteins which have mammalian orthologs, and are directly related to early mammalian development.