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

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.