SUMMARY The discovery of RNA interference and related small RNA pathways has revolutionized molecular biology and bears potential for new generations of therapeutics. Endogenous small RNAs such as microRNAs (miRNAs) collectively regulate the majority of the human transcriptome, and control important aspects of spatio-temporal development. Dysregulation of the miRNA pathway therefore directly contributes to developmental defects and tumorigenesis. Another group of small RNAs call Piwi-interacting RNAs (piRNAs) are often active during germline development, and preserve genome integrity by silencing selfish elements such as transposons. Despite of the progress made on understanding small RNA pathways, we still lack a clear picture of how each step of the pathway is carried out. Biochemical and structural characterization of the molecular machineries that promote these steps will represent major advances for the field. I have a long-standing interest in small RNA pathways. During my graduate studies at Princeton University with Dr. Laura Landweber, which were funded by a pre-doctoral fellowship by the Department of Defense Breast Cancer Research Program, I uncovered an unknown function for piRNAs to protect DNA against loss during genome rearrangement in Oxytricha. This was one of the first studies to show “self” recognition by piRNAs, and overturned the dogma that piRNAs only target RNA for repression or DNA for deletion. Then, as a post-doctoral fellow in the laboratory of Dr. David Bartel at the Whitehead Institute, I studied the miRNA pathway, which is the small RNA pathway that dominates in most human cells. Specifically, I identified determinants of primary miRNAs (pri-miRNAs, the precursors of miRNAs) that allow for their efficient processing by the Microprocessor complex, and demonstrated rational, de novo design of artificial miRNA genes. This work was funded by the Damon Runyon Cancer Research fellowship. My long-term goal is to understand how RNA-protein interactions work, with a special focus on small RNA pathways. I would like to pursue this exciting topic as the leader of a research group in an academic institution. To achieve this goal, the overall objectives of this application are to obtain training in protein biochemistry and single-particle cryo-electron microscopy (cryo-EM). This valuable training and experience will lay a solid foundation for me to launch my independent research on RNA-protein complexes. The work proposed here comprises two aims. In Aim 1, which will be completed during the mentored phase, I will leverage my knowledge of pri-miRNAs, the Microprocessor substrate, and harness recent development of cryo-EM, to solve the structure of Microprocessor bound with pri-miRNA, which will provide critical insights into how this complex recognizes its substrate and accurately positions the cleavage. I have already demonstrated that a novel, RNA-based purification strategy allowed the assembly of homogeneous Microprocessor ...