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

Abstract

Background: Ribose 2'-O-methylation, the most common nucleotide modification in mammalian rRNA, is directed by the C/D box small nucleolar RNAs (snoRNAs). Thus far, more than fifty putative human rRNA methylation guide snoRNAs have been identified. For nine of these snoRNAs, the respective ribose methylations in human 28S rRNA have been only presumptive.

Results: 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.

Conclusions: 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.

Figure 1

Location of novel 2'-O-methylated nucleotides in human 28S rRNA using reverse transcription of partially hydrolyzed rRNA and reverse transcription of rRNA at low dNTP concentrations. The positions of novel 2'-O-methylated nucleotides are shown in bold together with the putative snoRNA guide in parenthesis. Three previously detected methylations Cm2838, Am3794 and Am3799 are also indicated. Lanes 0', 30', 45' and 60' show primer extension products of partially hydrolyzed 28S rRNA, where the hydrolysis time is 0, 30, 45 and 60 min, respectively. Lanes, labeled 1 mM, 0.02 mM and 0.002 mM represent reverse transcription products of 28S rRNA at the indicated dNTP concentrations. Lanes A, C, G and T contain dideoxy sequencing ladders. Note that primer extension products generated with 5'-end-phosphorylated oligodeoxynucleotide primers migrate about 1 nucleotide farther than the respective sequencing reaction products produced with oligodeoxynucleotide primers bearing a hydroxyl group at the 5' end and terminating with dideoxynucleotides.