Understanding Recognition of Primary MicroRNAs by Human Drosha/DGCR8

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by suppressing messenger RNAs. The RNase Drosha, as part of the Microprocessor complex, performs the initial processing step in miRNA biogenesis. Drosha, in complex with its partner protein DGCR8, must distinguish its RNA substrates (called primary microRNAs) and identify the proper positions within these substrates to perform ribonuclease activity. My thesis work focuses on how the Drosha/DGCR8 complex recognizes primary microRNA structures and determines their processing sites, thereby regulating the initiation of microRNA biogenesis. Past biochemical and structural studies have left numerous unanswered questions about pri-miRNA processing. Using an in vitro system with human Drosha and DGCR8, I pinpointed the role of heme binding in DGCR8-mediated regulation of pri-miRNA processing, and revealed that heme activates DGCR8 to preferentially bind terminal loop structures, thereby orienting Drosha at the proper position for productive processing. Drosha tends to favor improper binding/processing sites on many pri-miRNAs, and heme-activated DGCR8 serves to correct this erroneous behavior and enable pri-miRNA processing at the appropriate site. Using information gained from these studies, I optimized a stable Drosha/DGCR8/pri-miRNA complex, which I then used as part of a collaboration to determine atomic structures through cryo-electron microscopy (cryo- EM). My cryo-EM structures revealed major new insights into how Drosha and DGCR8 ensure that pri-miRNAs possess the requisite structural features. A rigid assembly of double-stranded RNA-binding domains (dsRBDs) establishes a measuring system for pri-miRNA stem length. In addition, two newly identified regions of Drosha, the Wedge and the Belt, clamp over the basal end of bound RNAs and check for structural features required for efficient processing. In conjunction with the heme-binding region, which identifies the terminal loop, these components work together to recognize pri-miRNAs and enable processing. Overall, my work reveals critical information about how Drosha and DGCR8 work together to identify pri-miRNAs.