title: miRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity.
abstract: microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO-miRNA targeting are only partially understood. Here we report a modified AGO HITS-CLIP strategy termed CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP, which enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ∼130,000 endogenous miRNA-target interactions in mouse brain and ∼40,000 in human hepatoma cells. Motif and structural analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences but divergent 3'-ends. This work provides a means for explicit biochemical identification of miRNA sites in vivo, leading to the discovery that miRNA 3'-end pairing is a general determinant of AGO binding specificity.
authors.affiliation: Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 226, New York, New York 10065, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Moore
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 10065, USA.|Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, 2650 Hvidovre, Denmark.|Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
authors.forename: Troels K H
authors.initials: TK
authors.lastname: Scheel
authors.affiliation: Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 226, New York, New York 10065, USA.|Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Luna
authors.affiliation: Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 226, New York, New York 10065, USA.|New York Genome Center, 101 Avenue of the Americas, New York, New York 10013, USA.
authors.forename: Christopher Y
authors.initials: CY
authors.lastname: Park
authors.affiliation: Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 226, New York, New York 10065, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Fak
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Eiko
authors.initials: E
authors.lastname: Nishiuchi
authors.affiliation: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Rice
authors.affiliation: Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 226, New York, New York 10065, USA.|New York Genome Center, 101 Avenue of the Americas, New York, New York 10013, USA.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Darnell
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000378:metabolism*; D020029:Base Pairing; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D045744:Cell Line, Tumor; D002678:Chimera / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D060565:Argonaute Proteins; D035683:MicroRNAs; D012333:RNA, Messenger