MicroRNAs (miRNAs) are small regulatory RNAs that play a crucial role in gene silencing. The biogenesis of miRNAs begins with the cleavage of primary microRNA (pri-miRNA) by Microprocessor (MP), a complex of DROSHA and DGCR8. While MP's canonical cleavage mechanism has been well established, the cleavage of many pri-miRNAs cannot be explained by this mechanism. In this study, by conducting high-throughput cleavage assays for over 262,000 randomized pri-miRNA variants, we uncovered a noncanonical cleavage mechanism of MP. We found that this noncanonical mechanism relies on the DROSHA dsRNA recognition sites (DRES) rather than the essential RNA or protein elements of the canonical mechanism. We found that the DRES-dependent mechanism is essential in C. elegans and conserved across animals...[ Read more ]

MicroRNAs (miRNAs) are small regulatory RNAs that play a crucial role in gene silencing. The biogenesis of miRNAs begins with the cleavage of primary microRNA (pri-miRNA) by Microprocessor (MP), a complex of DROSHA and DGCR8. While MP's canonical cleavage mechanism has been well established, the cleavage of many pri-miRNAs cannot be explained by this mechanism. In this study, by conducting high-throughput cleavage assays for over 262,000 randomized pri-miRNA variants, we uncovered a noncanonical cleavage mechanism of MP. We found that this noncanonical mechanism relies on the DROSHA dsRNA recognition sites (DRES) rather than the essential RNA or protein elements of the canonical mechanism. We found that the DRES-dependent mechanism is essential in C. elegans and conserved across animals. This noncanonical mechanism also reveals how MP cleaves various pri-miRNAs, which cannot be explained by the canonical mechanism, and suggests that MP might utilize a broader class of substrates and have wider cellular activities.