title: The ribosome assembly factor Nep1 responsible for Bowen-Conradi syndrome is a pseudouridine-N1-specific methyltransferase.
abstract: Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen-Conradi syndrome-a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910-921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Psi) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Psi in eukaryotic 18S rRNAs.
authors.affiliation: Institut für Molekulare Biowissenschaften, Johann-Wolfgang-Goethe-Universität, 60438 Frankfurt/M., Germany.
authors.forename: Jan Philip
authors.initials: JP
authors.lastname: Wurm
authors.forename: Britta
authors.initials: B
authors.lastname: Meyer
authors.forename: Ute
authors.initials: U
authors.lastname: Bahr
authors.forename: Martin
authors.initials: M
authors.lastname: Held
authors.forename: Olga
authors.initials: O
authors.lastname: Frolow
authors.forename: Peter
authors.initials: P
authors.lastname: Kötter
authors.forename: Joachim W
authors.initials: JW
authors.lastname: Engels
authors.forename: Alexander
authors.initials: A
authors.lastname: Heckel
authors.forename: Michael
authors.initials: M
authors.lastname: Karas
authors.forename: Karl-Dieter
authors.initials: KD
authors.lastname: Entian
authors.forename: Jens
authors.initials: J
authors.lastname: Wöhnert
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D006801:Humans; D017015:Methanococcales / Q000201:enzymology* / Q000235:genetics; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D011560:Pseudouridine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020798:Two-Hybrid System Techniques
chemical_list: D019843:Archaeal Proteins; D009687:Nuclear Proteins; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; C013608:1-methylpseudouridine; D011560:Pseudouridine; C549212:EMG1 protein, human; D008780:Methyltransferases