The complete chemical structure of Saccharomyces cerevisiae rRNA: partial pseudouridylation of U2345 in 25S rRNA by snoRNA snR9.
We present the complete chemical structures of the rRNAs from the eukaryotic model organism, Saccharomyces cerevisiae The final structures, as determined with mass spectrometry-based methodology that includes a stable isotope-labelled, non-modified reference RNA, contain 112 sites with 12 different post-transcriptional modifications, including a previously unidentified pseudouridine at position 2345 in 25S rRNA. Quantitative mass spectrometry-based stoichiometric analysis of the different modifications at each site indicated that 94 sites were almost fully modified, whereas the remaining 18 sites were modified to a lesser extent. Superimposed three-dimensional modification maps for S. cerevisiae and Schizosaccharomyces pombe rRNAs confirmed that most of the modified nucleotides are located in functionally important interior regions of the ribosomes. We identified snR9 as the snoRNA responsible for pseudouridylation of U2345 and showed that this pseudouridylation occurs co-transcriptionally and competitively with 2'-O-methylation of U2345. This study ends the uncertainty concerning whether all modified nucleotides in S. cerevisiae rRNAs have been identified and provides a resource for future structural, functional and biogenesis studies of the eukaryotic ribosome.
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, Japan Science and Technology Agency, Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan mango@tmu.ac.jp.
Masato
M
Taoka
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, Japan Science and Technology Agency, Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan.
Yuko
Y
Nobe
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, Japan Science and Technology Agency, Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan.
Yuka
Y
Yamaki
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, Japan Science and Technology Agency, Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan.
Yoshio
Y
Yamauchi
Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 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.
Hideaki
H
Ishikawa
Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 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.
Nobuhiro
N
Takahashi
Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 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.
Hiroshi
H
Nakayama
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, Japan Science and Technology Agency, Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan isobe-toshiaki@tmu.ac.jp.
Toshiaki
T
Isobe
D001482:Base Composition; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D008745:Methylation; D008958:Models, Molecular; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics*; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000235:genetics*; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D012331:RNA, Fungal; D012335:RNA, Ribosomal; D020537:RNA, Small Nucleolar; C065039:RNA, ribosomal, 25S