Ribose methylation of eukaryotic rRNA is directed by box C/D small nucleolar RNAs (snoRNAs), which pinpoint the nucleotide to be methylated in specific position within the rRNA sequence. Here, we report the identification of a novel double-guide C/D box snoRNA termed snR88 that directs methylation of two previously undetermined sites in 25S rRNA from the fission yeast. Knockout of the predicted TATA box of the snR88 gene resulted in the complete blocking of its expression, showing that snR88 is an independently transcribed gene and dispensable for yeast viability. The depletion of snR88 abolished 255 rRNA methylation at U2304 and U2497 simultaneously. Interestingly, an unusual pause of reverse transcription at U2495 was observed, which implies an unknown structure of 25S rRNA related to ribose methylation at U2497 in the fission yeast. (c) 2007 Elsevier Inc. All rights reserved.