title: Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences.
abstract: 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 approximately 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.
authors.affiliation: Center for Virology and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zeng
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Cullen
mesh_terms: D001483:Base Sequence; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D035683:MicroRNAs / Q000737:chemistry*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000502:physiology*; D014162:Transfection
chemical_list: D035683:MicroRNAs; D012313:RNA; D005982:Glutathione Transferase; C517918:DROSHA protein, human; D043244:Ribonuclease III