Project Summary Non-coding RNAs are key regulators of diverse biological processes in eukaryotes. MicroRNAs are a family of small non-coding RNAs that post-transcriptionally regulate gene expression in a sequence-dependent manner. Approximately 1,000 human microRNAs appear to control the expression of more than half of all human messenger RNAs. Deviations from homeostatic microRNA expression levels, either reduced or enhanced expression, have been linked to cancers, diabetes, heart and neurodegenerative diseases, among others. To maintain proper microRNA expression levels, eukaryotic cells must tightly control the enzymatic processing of primary and precursor microRNA elements. However, the molecular determinants underlying this strict regulation of microRNA biogenesis are not fully understood. In this proposal we will explore both cis (RNA structure) and trans (protein binding partners) regulators of microRNAs biogenesis. In Research Area 1, we will explore the post-transcriptional regulation of members of the let-7 family of microRNAs. The processing of approximately half of the let-7 microRNAs is mediated by a protein partner, Lin28. The other members of this family are regulated by other, often unknown factors. We will determine the extent to which RNA modification, RNA structure, and RNA dynamics can serve as regulatory triggers to control the processing of these other family members. In Research Area 2, we will examine the differential processing of oncomiR-1, a polycistronic primary microRNA that is enriched in many cancers. We will conduct in cell chemical probing studies, identify associated cellular proteins, and elucidate a structural switch that controls processing of a subdomain. Our long-term goal is to determine the tertiary structure of oncomiR-1 alone and in complex with regulatory proteins. Collectively, these studies will help elucidate the molecular determinants underlying the highly-regulated production of microRNAs. RNA structures remain underdetermined relative to proteins, leading to an asymmetry in our mechanistic understanding of RNA folding and function. We expect that the methodology and approach developed in these studies will be broadly applicable to studies of other regulatory RNAs and protein-RNA complexes. This enhanced structural insight will inform on how RNA structure can directly regulate biological activity and will pave the way for the development of novel RNA-targeted therapeutics.