The high-throughput cleavage assays on 137 C.elegans pri-miRNAs

Microprocessor (MP), cleaving primary microRNAs (pri-miRNAs) to initiate the biogenesis of thousands of miRNAs, was discovered in animals in 2004. Understanding the molecular mechanism of MP is critical for interpreting the gene-silencing roles of miRNAs in various cellular processes and human diseases. Though the molecular mechanism of human MP (hMP) has been comprehensively investigated for nearly two decades and is well understood, that of Caenorhabditis elegans MP (cMP, cDrosha-Pasha complex) is still unknown. In this study, we revealed a comprehensive molecular mechanism of cMP, distinctively from that of hMP. In the proposed mechanism, cDrosha and Pasha (DGCR8 in humans) measure ~16 bp and ~25 bp of the pri-miRNA stem, respectively, and they are coordinated in determining cleavage sites of cMP in pri-miRNAs. In addition, we also demonstrated the molecular basis for their substrate measurement. Our findings illustrate an unknown molecular mechanism of cMP, clarify differences between the mechanism of hMP and cMP, and provide a foundation for understanding multiple mechanisms regulating miRNA expression acting on cMP. Overall design: The high-throughput cleavage assays for cDrosha, cMP WT and cMP mutants with 137 C.elegans pri-miRNAs