Structural and functional studies of Bud23–Trm112 reveal 18S rRNA N⁷-G1575 methylation occurs on late 40S precursor ribosomes

The eukaryotic small ribosomal subunit carries only four ribosomal (r) RNA methylated bases, all close to important functional sites. N ⁷-methylguanosine (m ⁷G) 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 ⁷G 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. Significance Ribosomes are essential cellular nanomachines responsible for all protein synthesis in vivo. Efficient and faithful ribosome biogenesis requires a plethora of assembly factors whose precise role and timing of action remains to be established. Here we determined the crystal structure of Bud23–Trm112, which is required for efficient pre-rRNA processing steps leading to 18S rRNA synthesis and methylation of 18S rRNA at position G1575. For the first time, to our knowledge, we identified where on Bud23–Trm112 the contacts with precursor ribosomes occur. We further report that the essential helicase Dhr1 interacts directly with Bud23–Trm112, proposing a concerted action of these proteins in ribosome assembly. Finally, we reveal that the methyltra