Noncanonical processing by animal Microprocessor.

Microprocessor (MP), DROSHA-DGCR8, processes primary miRNA transcripts (pri-miRNAs) to initiate miRNA biogenesis. The canonical cleavage mechanism of MP has been extensively investigated and comprehensively validated for two decades. However, this canonical mechanism cannot account for the processing of certain pri-miRNAs in animals. In this study, by conducting high-throughput pri-miRNA cleavage assays for approximately 260,000 pri-miRNA sequences, we discovered and comprehensively characterized a noncanonical cleavage mechanism of MP. This noncanonical mechanism does not need several RNA and protein elements essential for the canonical mechanism; instead, it utilizes previously unrecognized DROSHA dsRNA recognition sites (DRESs). Interestingly, the noncanonical mechanism is conserved across animals and plays a particularly significant role in C. elegans. Our established noncanonical mechanism elucidates MP cleavage in numerous RNA substrates unaccounted for by the canonical mechanism in animals. This study suggests a broader substrate repertoire of animal MPs and an expanded regulatory landscape for miRNA biogenesis.

Dissection of the Caenorhabditis elegans Microprocessor.

Microprocessor (MP) is a complex involved in initiating the biogenesis of microRNAs (miRNAs) by cleaving primary microRNAs (pri-miRNAs). miRNAs are small single-stranded RNAs that play a key role in the post-transcriptional regulation of gene expression. Thus, understanding the molecular mechanism of MP is critical for interpreting the roles of miRNAs in normal cellular processes and during the onset of various diseases. MP comprises a ribonuclease enzyme, DROSHA, and a dimeric RNA-binding protein, which is called DGCR8 in humans and Pasha in Caenorhabditis elegans. DROSHA cleaves stem-loop structures located within pri-miRNAs to generate pre-miRNAs. Although the molecular mechanism of human MP (hMP; hDROSHA-DGCR8) is well understood, that of Caenorhabditis elegans MP (cMP; cDrosha-Pasha) is still largely unknown. Here, we reveal the molecular mechanism of cMP and show that it is distinct from that of hMP. We demonstrate that cDrosha and Pasha measure ∼16 and ∼25 bp along a pri-miRNA stem, respectively, and they work together to determine the site of cMP cleavage in pri-miRNAs. We also demonstrate the molecular basis for their substrate measurement. Thus, our findings reveal a previously unknown molecular mechanism of cMP; demonstrate the differences between the mechanisms of hMP and cMP; and provide a foundation for revealing the mechanisms regulating miRNA expression in different animal species.

The Microprocessor complex that initiates miRNA biogenesis was discovered in animals in 2004. However, the molecular mechanism of C. elegans Microprocessor (cMP) has remained elusive since its discovery 18 years ago. In this study, we revealed the unique molecular mechanism of cMP by conducting high-throughput pri-miRNA cleavage assays. We demonstrated that cMP, consisting of cDrosha and Pasha, each can measure the stem lengths of pri-miRNAs. cDrosha measures ∼16 bp of the lower stem length, whereas Pasha measures ∼25 bp of the upper stem in pri-miRNAs. In addition, we identified the cleavage sites and cleavage efficiency of cMP in C. elegans pri-miRNAs. These results will be helpful for future studies of miRNA biogenesis in C. elegans.