Locations of several novel 2'-O-methylated nucleotides in human 28S rRNA

In this study, the methylation state of human 28S rRNA in the positions predicted by the snoRNAs U21, U26, U31, U48, U50, U73, U74, U80 and U81 was assessed using reverse transcription-based methods and several novel 2'-O-methylations were localized.Seven novel ribose 2'-O-methylated residues (Am389, Am391, Gm1604, Gm1739, Gm2853, Cm3810, Gm4156, predicted by snoRNAs U26, U81, U80, U73, U50, U74 and U31, respectively) have been localized in human 28S rRNA. The total number of 2'-O-methylations in human rRNA is not yet known.Ribose 2'-O-methylation is the most common nucleotide modification in mammalian rRNA. In human rRNA, localization of out of ≥ 100 2'-O-methylated residues has been reported (reviewed in [1]). Most of these ribose methylated nucleotides have been identified from RNase T1 and/or RNase A fingerprints. The remaining 12 or so that unplaced 2'-O-methylated nucleotides, all located in 28S rRNA, typically occurred in very short RNase T1 products; therefore, the precise positions of these 2'-O-methylations remained undetermined with this technique. In addition, 4 of 52 identified 28S rRNA ribose methylations have been mapped to within only two to four nucleotides [1].Over the past few years, it has become clear that selection of rRNA residues for ribose 2'-O-methylation is directed by the C/D box small nucleolar RNAs (snoRNAs). These snoRNAs contain common features including one or two 10–21 nucleotide long antisense elements complementary to mature rRNA and conserved sequence motifs called boxes C, D, C' and D'. When guiding 2'-O-methylation, the antisense element of a C/D box snoRNA base pairs with the corresponding region in pre-rRNA. Invariably, the rRNA residue base-paired to the fifth nucleotide upstream of the D or D' box becomes methylated in the rRNA [2,3]. The majority of the identified human C/D box snoRNAs are associated with known 2'-O-methylated nucleotides of either 18S or 28S rRNA (reviewed in [4]). However, there are also a number of pote