Much of eukaryotic gene regulation occurs post-transcriptionally, through differential mRNA stability and/or translational efficiency. The researc of this proposal seeks to answer fundamental questions within three interrelated areas of post-transcriptional gene control: microRNAs, RNA interference, and mRNA poly (A) tails. MicroRNAs (miRNAs) are ~22-nt RNAs that pair to mRNAs to direct their destabilization and translational repression. More than 600 miRNA genes have been identified in humans, and because most human genes are conserved targets of miRNAs, it is no surprise that miRNAs play important roles in mammalian development and human diseases, including viral infections and cancers. Molecular, computational, and structural approaches will be used to determine 1) how the microRNA-biogenesis machinery recognizes the cellular transcripts that are to be processed into microRNAs, 2) the biochemical basis of miRNA-target recognition and improved methods for predicting the most repressed targets, 3) the reason that mRNAs from reporter genes are repressed differently than those from endogenous genes, and 4) the mechanism and the biological function of the regulation of a miRNA by a long noncoding RNA. Results of these studies are expected to enhance the fundamental understanding of this important class of gene-regulatory molecules and provide resources helpful for many biologists, including those studying the roles of miRNAs in human diseases. RNA interference (RNAi) is a gene-regulatory pathway that many eukaryotic species use to silence transposons and viruses. In this pathway, short interfering RNAs (siRNAs) resembling miRNAs are loaded into Argonaute, which is an effector protein that cleaves transcripts with extensive complementarity to the siRNA. Genetic, structural, biochemical, and molecular approaches will be used to 1) identify and study additional proteins required for efficient RNAi in yeast, 2) determine how the siRNA-Argonaute complex forms and how it recognizes mRNA targets, and 3) investigate the unusual activities of zebrafish Argonaute. Results are expected to provide mechanistic insight into this gene-silencing pathway fundamental for defending many eukaryotic species against transposons and viruses, with practical implications for biologists using this pathway to study gene function, as well as those harnessing it to treat patients. mRNA poly (A) tails are important for mRNA stability and translational efficiency, and metazoan miRNAs usually act by recruiting enzymes that shortening poly (A) tails. The relationship between poly (A)-tail length and translational efficiency changes as the embryo develops. Molecular, computational, biochemical, and genetic approaches will be used to determine how coupling between tail length and translational efficiency is established before gastrulation and why it disappears after gastrulation. Results are expected to provide fundamental insight into translational control and embryonic development, with potential implications for human fertility, developmental defects, or other diseases. OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015) Page Continuation Format Page