Efficient processing of primary microRNA hairpins by drosha requires flanking nonstructured RNA sequences

Drosha is a member of the ribonuclease ( RNase) III family that selectively processes RNAs with prominent double-stranded features. Drosha plays a key role in the generation of precursor microRNAs from primary microRNA (pri-miRNA) transcripts in animal cells, yet how Drosha recognizes its RNA substrates remains incompletely understood. Previous studies have indicated that, within the context of a larger pri-miRNA, an similar to 80-nucleotide-long RNA hairpin structure is necessary for processing by Drosha. Here, by performing in vitro Drosha processing reactions with RNA substrates of various sizes and structures, we show that Drosha function also requires single-stranded RNA extensions located outside the pri-miRNA hairpin. The sequence of these RNA extensions was largely unimportant, but a strong secondary structure within the extension or a blunt-ended pri-miRNA hairpin blocked Drosha cleavage. The requirement for single-stranded extensions on the pri-miRNA hairpin substrate for Drosha processing is currently unique among the RNase III enzymes.