title: Structural and functional studies of Bud23-Trm112 reveal 18S rRNA N7-G1575 methylation occurs on late 40S precursor ribosomes.
abstract: The eukaryotic small ribosomal subunit carries only four ribosomal (r) RNA methylated bases, all close to important functional sites. N(7)-methylguanosine (m(7)G) introduced at position 1575 on 18S rRNA by Bud23-Trm112 is at a ridge forming a steric block between P- and E-site tRNAs. Here we report atomic resolution structures of Bud23-Trm112 in the apo and S-adenosyl-L-methionine (SAM)-bound forms. Bud23 and Trm112 interact through formation of a β-zipper involving main-chain atoms, burying an important hydrophobic surface and stabilizing the complex. The structures revealed that the coactivator Trm112 undergoes an induced fit to accommodate its methyltransferase (MTase) partner. We report important structural similarity between the active sites of Bud23 and Coffea canephora xanthosine MTase, leading us to propose and validate experimentally a model for G1575 coordination. We identify Bud23 residues important for Bud23-Trm112 complex formation and recruitment to pre-ribosomes. We report that though Bud23-Trm112 binds precursor ribosomes at an early nucleolar stage, m(7)G methylation occurs at a late step of small subunit biogenesis, implying specifically delayed catalytic activation. Finally, we show that Bud23-Trm112 interacts directly with the box C/D snoRNA U3-associated DEAH RNA helicase Dhr1 supposedly involved in central pseudoknot formation; this suggests that Bud23-Trm112 might also contribute to controlling formation of this irreversible and dramatic structural reorganization essential to overall folding of small subunit rRNA. Our study contributes important new elements to our understanding of key molecular aspects of human ribosomopathy syndromes associated with WBSCR22 (human Bud23) malfunction.
authors.affiliation: Laboratoire de Biochimie, CNRS UMR 7654, Ecole Polytechnique, F-91128 Palaiseau Cedex, France;
authors.forename: Juliette
authors.initials: J
authors.lastname: Létoquart
authors.affiliation: CNRS FRE3630 (affiliated with Université Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris F-75005, France;
authors.forename: Emmeline
authors.initials: E
authors.lastname: Huvelle
authors.affiliation: Center for Microscopy and Molecular Imaging, B-6041 Charleroi-Gosselies, Belgium; and.
authors.forename: Ludivine
authors.initials: L
authors.lastname: Wacheul
authors.affiliation: Laboratoire de Biochimie, CNRS UMR 7654, Ecole Polytechnique, F-91128 Palaiseau Cedex, France;
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Bourgeois
authors.affiliation: Center for Microscopy and Molecular Imaging, B-6041 Charleroi-Gosselies, Belgium; and.
authors.forename: Christiane
authors.initials: C
authors.lastname: Zorbas
authors.affiliation: Laboratoire de Biochimie, CNRS UMR 7654, Ecole Polytechnique, F-91128 Palaiseau Cedex, France; denis.lafontaine@ulb.ac.be marc.graille@polytechnique.edu valerie.heurgue@ibpc.fr.
authors.forename: Marc
authors.initials: M
authors.lastname: Graille
authors.affiliation: CNRS FRE3630 (affiliated with Université Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris F-75005, France; denis.lafontaine@ulb.ac.be marc.graille@polytechnique.edu valerie.heurgue@ibpc.fr.
authors.forename: Valérie
authors.initials: V
authors.lastname: Heurgué-Hamard
authors.affiliation: Center for Microscopy and Molecular Imaging, B-6041 Charleroi-Gosselies, Belgium; and RNA Molecular Biology, Fonds de la Recherche Scientifique, Université Libre de Bruxelles, B-6041 Charleroi-Gosselies, Belgium denis.lafontaine@ulb.ac.be marc.graille@polytechnique.edu valerie.heurgue@ibpc.fr.
authors.forename: Denis L J
authors.initials: DL
authors.lastname: Lafontaine
mesh_terms: D002384:Catalysis; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D012337:RNA, Ribosomal, 18S / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012337:RNA, Ribosomal, 18S; D029701:Saccharomyces cerevisiae Proteins; C578381:Bud23 protein, S cerevisiae; D008780:Methyltransferases; D012359:tRNA Methyltransferases; C502205:Trm112 protein, S cerevisiae
keywords: S-adenosyl-l-methionine; methyltransferase; rRNA modifying enzyme; ribosome synthesis; translation