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J. Biol. Chem., Vol. 266, Issue 36, 24385-24389, 12, 1991

A nucleolar 2'-O-methyltransferase. Specificity and evidence for its role in the methylation of mouse 28 S precursor ribosomal RNA

DM Segal and DC Eichler
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612.

Methylation of ribose moieties appears to be an essential post- transcriptional event in ribosomal RNA maturation. Although the sites of ribose methylation have been identified, the components involved in the 2'-O-methylation of precursor ribosomal RNA in mammalian cells have not yet been elucidated. To investigate the involvement of a recently isolated nucleolar 2'-O-methyltransferase in this process, an in vitro synthesized 28 S rRNA transcript containing a unique tandem triple 2'-O- methylated ribose site was used as a substrate. Activity assays demonstrated that this transcript served as a substrate for the nucleolar 2'-O-methyltransferase. The distribution of incorporated methyl groups was determined by hydrolyzing the 2'-O-methylated transcript with RNase followed by chromatography of the digested products on an anion-exchange high performance liquid chromatography column. Results showed one unique RNase-resistant 2'-O-methylated product, a tetramer. The position of the tetrameric sequence in the 28 S rRNA transcript was determined using RNase protection analysis which mapped the methylations to a 20-nucleotide region spanning the unique tandem triple 2'-O-methylated ribose site in 28 S rRNA. To confirm the absolute specificity of methylation, direct sequence analysis was carried out on the tandem triple 2'-O-methylated tetramer. The sequence determined for the tetramer, AmGmCmA, corresponded exactly with that reported from in vivo studies. These findings demonstrate that the purified nucleolar 2'-O-methyltransferase can accurately methylate at a specific site of an in vitro derived preribosomal RNA transcript and support the proposed involvement of this nucleolar enzyme in ribosomal RNA maturation.
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