Project Summary/Abstract (30 lines) MicroRNAs (miRNAs) constitute a large family of short, non-coding, regulatory RNAs that modulate protein expression. Abnormal miRNA levels are associated with many diseases, including developmental defects and various cancers. To generate functional miRNAs, primary transcripts (pri-miRNAs) generally need to be first cleaved by an RNaseIII, Drosha. This critical step of miRNA biogenesis needs to be controlled, in both accuracy and efficiency, to maintain proper gene regulation. The processing enzyme Drosha requires its partner protein, DGCR8, for protein stability and substrate specificity. A Drosha molecule binds homo-dimeric DGCR8 to carry out pri-miRNA processing, but higher-order complexes may also form because clustering of pri-miRNAs in the genome enhances processing. Our recent groundbreaking cryo-electron microscopy (cryo- EM) structures provide atomic models of the Microprocessor-pri-miRNA complex in action. Elucidating how the proteins are organized around the RNA stem-loop also revealed how each distal end (basal or apical) is independently recognized but also linked to each other via a molecular ruler connecting the detection modules. The proposed research builds on our previous successes, as the structural framework will enable us to gain novel fundamental insights into how the macromolecular recognition is accomplished. Our overall goal is to understand the recognition of pri-miRNAs by Microprocessor at the molecular level. We hypothesize that RNA structural features and context-dependent sequence preferences dictate the processing fate of each individual pri-miRNA. We will dissect how the recognition is accomplished at each of the basal and apical junctions of pri- miRNAs. We will also investigate how diverse RNA sequences and structures from different pri-miRNAs affect proper recognition at each junction, to reveal the plasticity of the multipart machinery. Our previous work has also left us poised to address urgent questions on how clustering of pri-miRNAs enhances processing by Microprocessor, which is crucial for our overall understanding of miRNA biogenesis and has profound implications for the interpretation of previous and future results in a wide variety of fields obtained by manipulation of miRNA genes. A better grasp of the core recognition mechanisms will help us explain unique targets such as clustered pri-miRNAs. Together, the proposed studies will provide a comprehensive understanding of processing and regulation of miRNAs, important regulators of gene expression. Our work on deciphering how structure affects RNA recognition is a fundamentally important question and likely be insightful for many processes involving structured RNAs beyond miRNAs.