Genome-wide Mapping of DROSHA Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates

MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here, we establish a protocol termed “formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq),” which allows identification of DROSHA cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of DROSHA substrates on non-miRNA loci, which may serve as cis-elements for DROSHA-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs. [Display omitted] •We develop fCLIP-seq, which identifies the substrates of DROSHA and their cleavage sites•Primary microRNA processing sites are mapped on a genomic scale•fCLIP-seq reveals widespread end modification and alternative processing of microRNA•Dozens of non-miRNA targets are discovered, suggesting noncanonical functions of DROSHA Kim et al. develop “fCLIP-seq” and use it to identify the targets of DROSHA, a microRNA-processing endonuclease. They map hundreds of primary microRNA cleavage sites and find evidence for microRNA end modification events and alternative DROSHA processing. They also discover many non-microRNA targets, suggesting non-canonical functions of DROSHA.