Abstract
Box C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2’-OH of RNA. The central ‘guide RNA’ has box C and D motifs at its ends, which are crucial for activity. Archaeal guide RNAs have a second box C’/D’ motif pair that is also essential for function. This second motif is poorly conserved in eukaryotes and its function is uncertain. Conflicting literature data report that eukaryotic box C’/D’ motifs do or do not bind proteins specialized to recognize box C/D-motifs and are or are not important for function. Despite this uncertainty, the architecture of eukaryotic 2’-O-methylation enzymes is thought to be similar to that of their archaeal counterpart. Here, we use biochemistry, X-ray crystallography and mutant analysis to demonstrate the absence of functional box C’/D’ motifs in more than 80% of yeast guide RNAs. We conclude that eukaryotic Box C/D RNPs have two non-symmetric protein assembly sites and that their three-dimensional architecture differs from that of archaeal 2’-O-methylation enzymes.
Highlights
Box C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2’-OH of RNA
The box D’ sequences corresponded to those annotated in the yeast snoRNA database UMass-Amherst[41]
Rather than study the structures of these RNAs in isolation, we tested whether they adopt the typical box C’/D’ kinked structure by assaying their ability to bind L7Ae. This protein has a very strong affinity for k-turn-like RNA structures[40] and binds box C/D sequences that deviate from the c onsensus[43,44], as well as RNAs that do not have a stable fold in isolation
Summary
Box C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2’-OH of RNA. Archaeal guide RNAs have similar additional motifs in the interior of the sequence, called boxes C’ and D’ (Fig. 1A)[25,26]; the motifs fold in either an internal k-turn or in a k-loop structure, depending on whether the kink is flanked by an RNA helix (stem I) or a loop[27]. These structures provide a second binding site for L7Ae, the archaeal orthologue of Snu[13]. The N-terminal domains of the eukaryotic Nop56–Nop[58] heterodimer bind the methyltransferase Nop[1] in yeast (fibrillarin in humans) and recruit it to the RNP (Supplementary Fig. 2)
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