Eukaryotic cells contain many fibrillarin-associated small nucleolar RNAs (snoRNAs) that possess long complementarities to mature rRNAs. Characterization of 21 novel antisense snoRNAs from human cells followed by genetic depletion and reconstitution studies on yeast U24 snoRNA provides evidence that this class of snoRNAs is required for site-specific 2'-O-methylation of preribosomal RNA (pre-rRNA). Antisense sno-RNAs function through direct base-pairing interactions with pre-rRNA. The antisense element, together with the D or D' box of the snoRNA, provide the information necessary to select the target nucleotide for the methyltransfer reaction. The conclusion that sno-RNAs function in covalent modification of the sugar moieties of ribonucleotides demonstrates that eukaryotic small nuclear RNAs have a more versatile cellular function than earlier anticipated.

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Site-specific 2Ј-O-ribose methylation of eukaryotic rRNAs is guided by small nucleolar RNAs (snoRNAs).The methylation guide snoRNAs carry long perfect complementaries to rRNAs. These antisense elements are located either in the 5Ј half or in the 3Ј end region of the snoRNA, and are followed by the conserved DЈ or D box motifs, respectively. An uninterrupted helix formed between the rRNA and the antisense element of the snoRNA, in conjunction with the adjacent DЈ or D box, constitute the recognition signal for the putative methyltransferase. Here, we have identified an additional essential box element common to methylation guide snoRNAs, termed the CЈ box. We show that the CЈ box functions in concert with the DЈ box and plays a crucial role in the methyltransfer reaction directed by the upstream antisense element and the DЈ box. We also show that an internal fragment of U24 methylation guide snoRNA, encompassing the upstream antisense element and the DЈ and CЈ box motifs, can support the site-specific methylation of rRNA. This strongly suggests that the C box of methylation guide snoRNAs plays an essential role in the methyltransfer reaction guided by the 3Ј-terminal antisense element and the D box of the snoRNA.