title: Two-step cleavage of hairpin RNA with 5' overhangs by human DICER.
abstract: BACKGROUND
  DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown.
  
  
  RESULTS
  In this study, we show that human recombinant DICER protein (rDICER) processes a hairpin RNA with 5' overhangs in vitro and generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage.
  
  
  CONCLUSIONS
  The two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures.
authors.affiliation: RIKEN Omics Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Yoshinari
authors.initials: Y
authors.lastname: Ando
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Maida
authors.forename: Ayako
authors.initials: A
authors.lastname: Morinaga
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Burroughs
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Kimura
authors.forename: Joe
authors.initials: J
authors.lastname: Chiba
authors.forename: Harukazu
authors.initials: H
authors.lastname: Suzuki
authors.forename: Kenkichi
authors.initials: K
authors.lastname: Masutomi
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Hayashizaki
mesh_terms: D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D035683:MicroRNAs; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases