Structural and functional studies of Bud23–Trm112 reveal 18S rRNA N ⁷ -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.ribosome synthesis | rRNA modifying enzyme | methyltransferase | translation | S-adenosyl-L-methionine

Utp14 Recruits and Activates the RNA Helicase Dhr1 To Undock U3 snoRNA from the Preribosome

“…9B). This suggests that Bud23 alone is not necessary for recruitment of Dhr1 to the preribosome, contrary to a recent report (21). Given that Utp14 also interacts with Dhr1, we asked if Utp14 is required for association of Dhr1 with the preribosome.…”

“…We took advantage of the slowgrowth defect of bud23 mutants as a genetic entry point to dissect a constellation of functionally linked proteins. We found that mutations in DHR1, UTP2, or UTP14 partially suppressed the growth defect and 40S assembly defect of bud23⌬ mutants (18,21,23). We also showed that Bud23 binds to the N-terminal extension of Dhr1, making Bud23 a candidate for recruiting or regulating Dhr1 (24).…”

“…Utp14 was initially characterized as a component of the SSU processome (8) and likely associates with the preribosome before Bud23 (23). Bud23 methylates G1575 in the 3= major head domain of the subunit (21,22) and presumably binds to its RNA target site only after it is properly folded but before A2 cleavage, as bud23⌬ mutants are defective for cleavage at this site (22). Both Utp14 and Bud23 remain associated with the subunit after A2 cleavage (23) and thus mark the transition from the 90S particle to the pre-40S particle.…”

“…The methyltransferase Bud23 modifies the guanosine base at position 1575 in 18S rRNA (21,22). Bud23 is recruited to the preribosome at a relatively late step in 40S biogenesis, before A2 cleavage, and remains associated with the pre-40S particle in the nucleus (21,23). We took advantage of the slowgrowth defect of bud23 mutants as a genetic entry point to dissect a constellation of functionally linked proteins.…”

Publisher's Note

“…It has now been well established both in yeast and human systems that, in some cases, enzymes bind to rRNA early in biogenesis, but do not actually catalyze a reaction until a later phase of maturation. 76,77 Similarly, in some cases enzymes modify RNAs in multiple classes, 78 so loss of function phenotypes must be analyzed with care. Thus it is crucial that, as researchers delve deeper into the functions of modifications in all RNA classes, the roles of the enzymes and the modifications themselves are disentangled.…”