A nucleolar 2'-O-methyltransferase. Specificity and evidence for its role in the methylation of mouse 28 S precursor ribosomal RNA.
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.
Segal
D M
DM
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612.
Eichler
D C
DC
D000818:Animals; D001483:Base Sequence; D002466:Cell Nucleolus / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012339:RNA, Ribosomal, 28S / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D012322:RNA Precursors; D012339:RNA, Ribosomal, 28S; D008780:Methyltransferases