title: An RNA-binding Protein, Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates.
abstract: Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function.
authors.affiliation: Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115; Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, Massachusetts 02115.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: O'Day
authors.affiliation: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, Massachusetts 02115.
authors.forename: Minh T N
authors.initials: MTN
authors.lastname: Le
authors.affiliation: Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Imai
authors.affiliation: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, Massachusetts 02115.
authors.forename: Shen Mynn
authors.initials: SM
authors.lastname: Tan
authors.affiliation: Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts 02115.
authors.forename: Rory
authors.initials: R
authors.lastname: Kirchner
authors.affiliation: Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Haribabu
authors.initials: H
authors.lastname: Arthanari
authors.affiliation: Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts 02115.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hofmann
authors.affiliation: Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115. Electronic address: Gerhard_wagner@hms.harvard.edu.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wagner
authors.affiliation: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, Massachusetts 02115. Electronic address: Judy.Lieberman@childrens.harvard.edu.
authors.forename: Judy
authors.initials: J
authors.lastname: Lieberman
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D054856:G-Quadruplexes; D006801:Humans; D008652:Mesoporphyrins / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: C488352:Lin-28 protein, mouse; C000715187:Lin28A protein, human; D008652:Mesoporphyrins; D035683:MicroRNAs; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C065420:N-methylmesoporphyrin IX
keywords: G-quartet; Lin28; N-methyl mesoporphyrin IX (NMM); cancer; embryonic stem cell; fluorescence; let-7; microRNA (miRNA); nuclear magnetic resonance (NMR)