title: Global identification of target recognition and cleavage by the Microprocessor in human ES cells.
abstract: The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3' overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells.
authors.affiliation: Department of Bioscience & Biotechnology, Sejong University, Seoul 143-747, Korea.
authors.forename: Youngmo
authors.initials: Y
authors.lastname: Seong
authors.affiliation: College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea.
authors.forename: Do-Hwan
authors.initials: DH
authors.lastname: Lim
authors.affiliation: Department of Food Science & Technology, Sejong University, Seoul 143-747, Korea.
authors.forename: Augustine
authors.initials: A
authors.lastname: Kim
authors.affiliation: Department of Internal Medicine, Korea University Guro Hospital, Seoul 152-703, Korea.
authors.forename: Jae Hong
authors.initials: JH
authors.lastname: Seo
authors.affiliation: College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea.
authors.forename: Young Sik
authors.initials: YS
authors.lastname: Lee
authors.affiliation: Department of Biomedical Sciences, Korea University, Seoul 136-705, Korea yngskwon@sejong.ac.kr.
authors.forename: Hoseok
authors.initials: H
authors.lastname: Song
authors.affiliation: Department of Bioscience & Biotechnology, Sejong University, Seoul 143-747, Korea yngskwon@sejong.ac.kr.
authors.forename: Young-Soo
authors.initials: YS
authors.lastname: Kwon
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000201:enzymology* / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D059367:RNA Cleavage; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism*; D017423:Sequence Analysis, RNA
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; C474043:DGCR8 protein, human; D035683:MicroRNAs; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C517918:DROSHA protein, human; D043244:Ribonuclease III