title: Eukaryotic rRNA Modification by Yeast 5-Methylcytosine-Methyltransferases and Human Proliferation-Associated Antigen p120.
abstract: Modified nucleotide 5-methylcytosine (m5C) is frequently present in various eukaryotic RNAs, including tRNAs, rRNAs and in other non-coding RNAs, as well as in mRNAs. RNA:m5C-methyltranferases (MTases) Nop2 from S. cerevisiae and human proliferation-associated nucleolar antigen p120 are both members of a protein family called Nop2/NSUN/NOL1. Protein p120 is well-known as a tumor marker which is over-expressed in various cancer tissues. Using a combination of RNA bisulfite sequencing and HPLC-MS/MS analysis, we demonstrated here that p120 displays an RNA:m5C- MTase activity, which restores m5C formation at position 2870 in domain V of 25S rRNA in a nop2Δ yeast strain. We also confirm that yeast proteins Nop2p and Rcm1p catalyze the formation of m5C in domains V and IV, respectively. In addition, we do not find any evidence of m5C residues in yeast 18S rRNA. We also performed functional complementation of Nop2-deficient yeasts by human p120 and studied the importance of different sequence and structural domains of Nop2 and p120 for yeast growth and m5C-MTase activity. Chimeric protein formed by Nop2 and p120 fragments revealed the importance of Nop2 N-terminal domain for correct protein localization and its cellular function. We also validated that the presence of Nop2, rather than the m5C modification in rRNA itself, is required for pre-rRNA processing. Our results corroborate that Nop2 belongs to the large family of pre-ribosomal proteins and possesses two related functions in pre-rRNA processing: as an essential factor for cleavages and m5C:RNA:modification. These results support the notion of quality control during ribosome synthesis by such modification enzymes.
authors.affiliation: Laboratoire IMoPA, UMR 7365 UL-CNRS, BioPole de UL, Vandoeuvre-les-Nancy, France.
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Bourgeois
authors.affiliation: Laboratoire IMoPA, UMR 7365 UL-CNRS, BioPole de UL, Vandoeuvre-les-Nancy, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Ney
authors.affiliation: EMBL Heidelberg, Meyerhofstraße 1, 69117, Heidelberg, Germany.
authors.forename: Imre
authors.initials: I
authors.lastname: Gaspar
authors.affiliation: Laboratoire IMoPA, UMR 7365 UL-CNRS, BioPole de UL, Vandoeuvre-les-Nancy, France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Aigueperse
authors.affiliation: Division of Epigenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schaefer
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Mainz, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kellner
authors.affiliation: Institute of Pharmacy and Biochemistry, Johannes Gutenberg-University Mainz, Mainz, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Helm
authors.affiliation: Laboratoire IMoPA, UMR 7365 UL-CNRS, BioPole de UL, Vandoeuvre-les-Nancy, France.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Motorin
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism*; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012335:RNA, Ribosomal / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009687:Nuclear Proteins; D012335:RNA, Ribosomal; D029701:Saccharomyces cerevisiae Proteins; C065039:RNA, ribosomal, 25S; D044503:5-Methylcytosine; D008780:Methyltransferases; C091276:NOP2 protein, S cerevisiae; C065028:NOP2 protein, human; D012359:tRNA Methyltransferases