Létoquart Juliette, Huvelle Emmeline, Wacheul Ludivine, Bourgeois Gabrielle, Zorbas Christiane, Graille Marc, Heurgué-Hamard Valérie, Lafontaine Denis L J
Laboratoire de Biochimie, CNRS UMR 7654, Ecole Polytechnique, F-91128 Palaiseau Cedex, France;
CNRS FRE3630 (affiliated with Université Paris Diderot, Sorbonne Paris Cité), Institut de Biologie Physico-Chimique, Paris F-75005, France;
Proc Natl Acad Sci U S A. 2014 Dec 23;111(51):E5518-26. doi: 10.1073/pnas.1413089111. Epub 2014 Dec 8.
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.
真核生物小核糖体亚基仅携带四个核糖体（r）RNA甲基化碱基，它们都靠近重要的功能位点。由Bud23-Trm112在18S rRNA的1575位引入的N（7）-甲基鸟苷（m（7）G）位于一个脊上，在P位点和E位点tRNA之间形成空间位阻。在此，我们报道了Bud23-Trm112的无配体形式和结合S-腺苷-L-甲硫氨酸（SAM）形式的原子分辨率结构。Bud23和Trm112通过形成涉及主链原子的β-拉链相互作用，掩埋了一个重要的疏水表面并稳定了复合物。这些结构表明，共激活因子Trm112经历了诱导契合以容纳其甲基转移酶（MTase）伙伴。我们报道了Bud23和咖啡黄嘌呤甲基转移酶的活性位点之间存在重要的结构相似性，这使我们提出并通过实验验证了G1575配位的模型。我们确定了对Bud23-Trm112复合物形成和募集到前核糖体很重要的Bud23残基。我们报道，尽管Bud23-Trm112在核仁早期阶段结合前体核糖体，但m（7）G甲基化发生在小亚基生物合成的后期步骤，这意味着催化激活特别延迟。最后，我们表明Bud23-Trm112直接与假定参与中央假结形成的盒C/D小核仁RNA U3相关的DEAH RNA解旋酶Dhr1相互作用；这表明Bud23-Trm112也可能有助于控制这种对小亚基rRNA整体折叠至关重要的不可逆且剧烈的结构重组的形成。我们的研究为我们理解与WBSCR22（人类Bud23）功能障碍相关的人类核糖体病综合征的关键分子方面贡献了重要的新元素。
