title: A mass spectrometry-based method for comprehensive quantitative determination of post-transcriptional RNA modifications: the complete chemical structure of Schizosaccharomyces pombe ribosomal RNAs.
abstract: We present a liquid chromatography-mass spectrometry (LC-MS)-based method for comprehensive quantitative identification of post-transcriptional modifications (PTMs) of RNA. We incorporated an in vitro-transcribed, heavy isotope-labeled reference RNA into a sample RNA solution, digested the mixture with a number of RNases and detected the post-transcriptionally modified oligonucleotides quantitatively based on shifts in retention time and the MS signal in subsequent LC-MS. This allowed the determination and quantitation of all PTMs in Schizosaccharomyces pombe ribosomal (r)RNAs and generated the first complete PTM maps of eukaryotic rRNAs at single-nucleotide resolution. There were 122 modified sites, most of which appear to locate at the interface of ribosomal subunits where translation takes place. We also identified PTMs at specific locations in rRNAs that were altered in response to growth conditions of yeast cells, suggesting that the cells coordinately regulate the modification levels of RNA.
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
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: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Hori
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Takeuchi
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: Shunpei
authors.initials: S
authors.lastname: Masaki
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: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan Biomolecular Characterization Team, RIKEN Center for SustainableResource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakayama
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: 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
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D012323:RNA Processing, Post-Transcriptional; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism
chemical_list: D012335:RNA, Ribosomal