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. Labelled 17-S and 26-S rRNA were prepared from cells that had been incubated with either [32P]phosphate, methionine. Ion-exchange resin chromatography of 0.3 M KOH or 1 M HCl hydrolysates and two-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. "-Methyladenine and N6,Nh-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 one 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 three times, respectively, in 26-S rRNA. 7-Methylguanine and 5-methylcytosine are only in submolar amounts in the 26-S and 17-S rRNA, respectively.There are 40 & 2 and 83 f 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, one and seven 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 HeLa cell rRNA.The methylation of specific parts of polynucleotide chains is one of the multiple molecular events altering the primary transcriptional product of ribosomal genes and leading to the formation of the mature cytoplasmic rRNA [l]. Initial reports on the methylation of Escherichia coli rRNA and animal cytoplasmic rRNA suggested that eukaryotic cytoplasmic rRNA and prokaryotic rRNA strongly differ with regard to their type of methylation, the former being essentially ribose-methylated and the latter base-methylated. Later, specific methylated bases were identified in E. coli [2], yeast [3] and HeLa cells [4-61. These data supported the rather new idea that base-methylation might be an ancestral process widely conserved in living cells whereas ribose-methylation might represent a less-primitive process that appeared essentially in eukaryotic cells during evolution [l].With regard to cytoplasmic rRNA of higher plants, only 2'-O-methylation had been investigated [7-91. There had been no report on their basemethylation. Additional data on plant cells were

Fibrillarin genes encode both a conserved nucleolar protein and a novel snoRNA involved in rRNA methylation in Arabidopsis thaliana

“…Thus methylation at Am2116 could either implicate another unidentified snoRNA or, alternatively, U60.1f/.2f could methylate both Gm2114 and Am2116. No other modified nucleotides have been mapped except for the three reported here, but early biochemical studies indicate that neighbouring 2'-O-methylated residues do occur in plant rRNAs (29).…”

“…These include U3, U14 and seven other box C/D and one box H/ACA snoRNAs described in Arabidopsis or maize (28). Many other snoRNAs should exist in plants since early biochemical studies indicate the presence of numerous modified nucleotides in plant rRNAs (29), but these have never by guest on May 7, 2018 http://www.jbc.org/ Downloaded from been mapped. In addition, many Arabidopsis genomic sequences displaying box C/D or H/ACA snoRNA features can be find in the databanks.…”

Plant dicistronic tRNA-snoRNA genes: a new mode of expression of the small nucleolar RNAs processed by RNase Z

“…In plants, early biochemical data revealed that rRNAs are highly modified implicating overall 120 2′‐ O ‐ribose methylated residues (Lau et al ., 1974; Cecchini and Miassod, 1979). The mechanisms guiding rRNA methylation depending on fibrillarin and C/D snoRNAs are conserved in plants (Barneche et al ., 2000).…”

Identification of six new box C/D snoRNA gene clusters from rice

“…Early studies revealed more than 120 2 0 -O-ribose methylation sites in the rRNAs of sycamore plant cell (23). Compared with other eukaryotic species, plants contain a number of 2 0 -O-ribose methylation sites in rRNAs (24,25).…”

“…Since the 1990s, numerous novel snoRNA homologues have been identified from various eukaryotes including vertebrate, invertebrates, yeast and protests, and even from archael organisms (15 -22). Early studies revealed more than 120 2 0 -O-ribose methylation sites in the rRNAs of sycamore plant cell (23). Compared with other eukaryotic species, plants contain a number of 2 0 -O-ribose methylation sites in rRNAs (24,25).…”