Studies on the Methylation of Cytoplasmic Ribosomal RNA from Cultured Higher Plant Cells

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Abstract
The methylation of cytoplasmic ribosomal RNA of cultured sycamore cells (Acer pseudoplatanus L.) was investigated. Labeled 17-S and 26-S rRNA were prepared from cells that had been incubated with either [32P]phosphate, [Me-3H]methionine or [Me-14C]methionine. Ion-exchange resin chromatography of 0.3 M KOH or 1 M HCl hydrolysates and 2-dimensional chromatographic analyses of phosphodiesterase plus phosphatase digests of 17-S and 26-S rRNA were performed. 17-S and 26-S rRNA contain 49 and 91 methyl groups per molecule, respectively. These values were verified in several ways. The high degree of methylation of sycamore rRNA, particularly for the 26-S rRNA, contrasts with the situation in all other investigated organisms. Several methylated bases were identified. 7-Methylguanine and 5-methylcytosine both occur in 17-S and 26-S rRNA. N6-methyladenine and N6,N6-dimethyladenine are restricted to the 17-S rRNA while 3-methyluracil and 1-methyladenine occur in the 26-S rRNA. One hypermodified uridine was also tentatively identified in the small rRNA. In 17-S rRNA, there is 1 copy of 7-methylguanine, N6-methyladenine and hypermodified uridine and two copies of N6,N6-dimethyladenine. 3-Methyluracil, 1-methyladenine and 5-methylcytosine occur twice, twice and 3 times, respectively, in 26-S rRNA. 7-Methylguanine and 6-methylcytosine are only in submolar amounts in the 26-S and 17-S rRNA, respectively. There are 40 .+-. 2 and 83 .+-. 3 2''-O-methylriboses per 17-S and 26-S rRNA molecule, respectively. In addition to the four 2''-O-methylnucleosides, one 2''-O-methylpseudouridine is present in the 17-S rRNA. Several lines of evidence argue for a non-random distribution of the methylriboses. In particular, 1 and 7 Nm-Nm-Np structures occur in the 17-S and 26-S rRNA, respectively. The data are discussed comparatively with the methylation pattern of Escherichia coli, yeast and [human cervical carcinoma] HeLa cell rRNA.