title: A mass spectrometry-based method for direct determination of pseudouridine in RNA.
abstract: Pseudouridine (5-ribosyluracil, Ψ) is the only 'mass-silent' nucleoside produced by post-transcriptional RNA modification. We describe here a novel mass spectrometry (MS)-based method for direct determination of Ψ in RNA. The method assigns a Ψ-containing nucleolytic RNA fragment by an accurate measurement of a signature doubly dehydrated nucleoside anion ([C9H7N2O4](1-),m/z207.04) produced by collision-induced dissociation MS, and it determines the Ψ-containing nucleotide sequence by pseudo-MS(3), i.e. in-source fragmentation followed by MS(2) By applying this method, we identified all of the known Ψs in the canonical human spliceosomal snRNAs and, unexpectedly, found two previously unknown Ψs in the U5 and U6 snRNAs. Because the method allows direct determination of Ψ in a subpicomole quantity of RNA, it will serve as a useful tool for the structure/function studies of a wide variety of non-coding RNAs.
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Yamauchi
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nobe
authors.affiliation: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan Department of Biotechnology, United Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Saiwai-cho 3-5-8, Fuchu-shi, Tokyo 183-8509, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Izumikawa
authors.affiliation: Thermo Fisher Scientific, 3-9 Moriya-cho, Kanagawa-ku, Yokohama-shi, Kanagawa 221-0022, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Higo
authors.affiliation: Thermo Fisher Scientific, 3-9 Moriya-cho, Kanagawa-ku, Yokohama-shi, Kanagawa 221-0022, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Yamagishi
authors.affiliation: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan Department of Biotechnology, United Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Saiwai-cho 3-5-8, Fuchu-shi, Tokyo 183-8509, Japan.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Takahashi
authors.affiliation: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakayama
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan isobe-toshiaki@tmu.ac.jp.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Isobe
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan mango@tmu.ac.jp.
authors.forename: Masato
authors.initials: M
authors.lastname: Taoka
mesh_terms: D001483:Base Sequence; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011560:Pseudouridine / Q000737:chemistry* / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D017381:Spliceosomes / Q000737:chemistry / Q000378:metabolism
chemical_list: D012342:RNA, Small Nuclear; C405485:U5 small nuclear RNA; C405486:U6 small nuclear RNA; D011560:Pseudouridine