Identification of the locations of the methyl groups in 18 S ribosomal RNA from Xenopus laevis and man

The 18 S ribosomal RNA from a variety of vertebrate species contains some 40 to 47 methyl groups. The majority of these are 2'-O-ribose substituents; the remaining few are on bases. Several lines of evidence have permitted the identification of the precise locations of the methyl groups in the primary structure of 18 S ribosomal RNA of Xenopus laevis and man. Digestion of RNA with T1 ribonuclease, followed by analysis of the methylated oligonucleotides yielded data on sequences immediately surrounding the methyl groups. Preparative hybridization of X. laevis 18 S ribosomal RNA restriction fragments of ribosomal DNA, followed by fingerprinting analysis on RNA recovered from the hybrids, allowed each methylated oligonucleotide to be mapped to a specific region within 18 S ribosomal RNA. The data on RNA oligonucleotides were correlated with Xenopus ribosomal DNA sequence data in the regions defined by the mapping experiments to identify the precise locations of most of the methyl groups in the X. laevis 18 S RNA sequence. The remaining uncertainties in Xenopus were solved with the aid of data from ribonuclease A fingerprints and, in a few instances, relevant oligonucleotide or sequence data from other laboratories. The locations of most of the methyl groups in human 18 S ribosomal RNA were deduced from the high degree of correspondence between methylated oligonucleotides from human and X. laevis 18 S RNA, together with knowledge of the human 18 S ribosomal DNA sequence. The remaining methylation sites in human 18 S RNA were located with assistance from relevant published comparative data. In the aligned sequences, human and other mammalian 18 S RNA are methylated at all the same positions as in X. laevis, and there are seven additional 2'-O-methylation sites in mammalian 18 S RNA. Further features of the methyl group distribution are briefly reviewed.     

Nucleotide sequence encoding the 5'' end of Xenopus laevis 18S rRNA.

We have sequenced a region of cloned Xenopus laevis ribosomal DNA encompassing the last 24 nucleotides of the external transcribed spacer and the first 275 nucleotides of the 18S gene. The start of the 18S gene was identified by correlating the results obtained from RNA hybridization and fingerprinting with the DNA sequence. This 5' region of 18S rRNA contains five 2'-O-methyl groups and at least six pseudouridine residues. Several of these modified nucleotides are clustered into a relatively short region from nucleotides 99-124. Nucleotides 227-250 constitute a distinctive sequence of 24 consecutive G and C residues. Comparison with the first 160 nucleotides of a yeast 18S gene (25) reveals three blocks of high sequence homology separated by two short tracts where homology is low or absent. The external transcribed spacer sequences diverge widely from within a few nucleotides of the start of the 18S gene.     

Human 18 S ribosomal RNA sequence inferred from DNA sequence. Variations in 18 S sequences and secondary modification patterns between vertebrates.

We have determined the DNA sequences encoding 18 S ribosomal RNA in man and in the frog, Xenopus borealis. We have also corrected the Xenopus laevis 18 S sequence: an A residue follows G-684 in the sequence. These and other available data provide a number of representative examples of variation in primary structure and secondary modification of 18 S ribosomal RNA between different groups of vertebrates. First, Xenopus laevis and Xenopus borealis 18 S ribosomal genes differ from each other by only two base substitutions, and we have found no evidence of intraspecies heterogeneity within the 18 S ribosomal DNA of Xenopus (in contrast to the Xenopus transcribed spacers). Second, the human 18 S sequence differs from that of Xenopus by approx. 6.5%. About 4% of the differences are single base changes; the remainder comprise insertions in the human sequence and other changes affecting several nucleotides. Most of these more extensive changes are clustered in a relatively short region between nucleotides 190 and 280 in the human sequence. Third, the human 18 S sequence differs from non-primate mammalian sequences by only about 1%. Fourth, nearly all of the 47 methyl groups in mammalian 18 S ribosomal RNA can be located in the sequence. The methyl group distribution corresponds closely to that in Xenopus, but there are several extra methyl groups in mammalian 18 S ribosomal RNA. Finally, minor revisions are made to the estimated numbers of pseudouridines in human and Xenopus 18 S ribosomal RNA.     

Pseudouridine distribution in mammalian 18 S ribosomal RNA. A major cluster in the central region of the molecule.

Human and rodent 18 S rRNA contain about 38 pseudouridine residues. By correlating RNA oligonucleotide data with complete sequence data derived from ribosomal DNA, 30 pseudouridine residues can be located in the RNA sequence, either exactly or to within two or three residues. Pseudouridine and 2'-O-methyl groups are interspersed throughout mammalian 18 S rRNA, but not in closely parallel fashion. Whereas the largest cluster of 2'-O-methyl groups is in the 5' one-third of the molecule, the greatest concentration of pseudouridine is in the central one-third of the molecule.     

The methylated nucleotide sequences in HELA cell ribosomal RNA and its precursors

The methylated nucleotide sequences in HeLa cell ribosomal RNA and its nucleolar precursors were examined by RNA fingerprinting and sequencing methods. 18 S RNA was found to contain approximately 46 methyl groups, 28 S RNA some 70 methyl groups and 5.8 S RNA one methyl group. Most methyl groups occur in different T₁ ribonuclease oligonucleotides, and most of these were recovered approximately once per molecule of 18 S or 28 S RNA. There are also, however, several multiply methylated oligonucleotides, a few short products that occur more than once and a few “fractional” products. The great majority of methylations occur at the level of 45 S RNA, but six further methylations occur late during the maturation of 18 S RNA, and one fractional one occurs during 28 S maturation. The transcribed spacer regions of the precursor molecules are unmethylated. Chemical analysis of the methylated components and sequences indicates that all except five “early” methylations are on ribose groups, the remaining five being on bases within the 28 S sequence. The late methylations are all on bases, four of those on 18 S RNA giving rise to the sequence, … Gpm₂^post6Apm₂^post6ApCp… The product, pCmpUp, previously reported by Choi &; Busch (1970) as being the 5′ end-group of rat hepatoma 28 S, 32 S and 45 S RNA, is not present in HeLa cell 28 S RNA or its precursors. Implications of this work are discussed.     

Nucleotide sequence of the 5'- and 3'- domains for rabbit 18S ribosomal RNA.

By direct RNA sequence analysis we have determined the primary structures of both the 5' and 3' domains for rabbit 18S ribosomal RNA. Purified 18S rRNA was labeled in vitro at either its 5' or 3' terminus with 32P, base-specifically fragmented enzymatically and chemically, and the resulting fragments electrophoretically fractionated by size in adjacent lanes of 140 cm long polyacrylamide sequencing gels run in 90% formamide. A phylogenetic comparison of both the mammalian 5' proximal 400 residues and the 3' distal 301 nucleotides with the previously determined yeast and Xenopus laevis 18S rRNA sequence shows extensive conservation interspersed with tracts having little homology. Clusters of G + C rich sequences are present within the mammalian 5' domain which are entirely absent in both the Xenopus laevis and yeast 18S rRNAs. Most base differences and insertions within the mammalian 18S rRNA when compared with yeast or Xenopus rRNA result in an increase in the G + C content of these regions. We have found nucleotide sequence analysis of the ribosomal RNA directly permits detection of both cistron heterogeneities and mapping of many of the modified bases.     

A new method for detecting sites of 2'-O-methylation in RNA molecules.

2'-O-methylation of eukaryotic ribosomal RNAs occurs in the cell nucleoli. At least 100 modification sites that are highly conserved among vertebrate rRNAs have been mapped. However, in part because of the insensitivity of current approaches, there are 2'-O-methylated sites that remain unidentified. We have developed an extremely sensitive method for detecting 2'-O-methylated residues that are predicted within a long RNA molecule. Utilizing RNase H cleavage directed by a 2'-O-methyl RNA-DNA chimeric oligonucleotide, this method has allowed identification of two methylated nucleotides, G1448 in Xenopus 18S rRNA and A394 in Xenopus 28S rRNA. The latter (A394 in 28S) had not been detected before. We have confirmed that the methylation at G1448 in 18S is dependent upon Xenopus U25 snoRNA and have demonstrated that the methylation at A394 in 28S requires U26 snoRNA. One advantage of this technique is that it can examine specific rRNA and precursor molecules. We show that about 30% of the 40S pre-rRNA has been methylated at these two sites and their methylation is complete at the stage of 20S (immediate precursor to 18S) and 32S (immediate precursor to 28S). We also show that methylation at these two sites is not essential for rRNA transport from the nucleus to the cytoplasm.     

Xenopus laevis 18S ribosomal RNA: experimental determination of secondary structural elements, and locations of methyl groups in the secondary structure model.

18S ribosomal RNA from X. laevis was subjected to partial digestion with ribonucleases A or T1 under a variety of conditions, and base-paired fragments were isolated. Sequence analysis of the fragments enabled five base-paired secondary structural elements of the 18S RNA to be established. Four of these elements (covering bases 221-256, 713-757, 1494-1555 and 1669-1779) confirm our previous secondary structure predictions, whereas the fifth (comprising bases 1103-1125) represents a phylogenetically conserved "switch" structure, which can also form in prokaryotic 16S RNA. The results are incorporated into a refined model of the 18S RNA secondary structure, which also includes the locations of the many methyl groups in X. laevis 18S RNA. In general the methyl groups occur in non-helical regions, at hairpin loop ends, or at helix boundaries and imperfections. One large cluster of 2'-O-methyl groups occurs in a region of complicated secondary structure in the 5'-one third of the molecule.     

Secondary structure maps of ribosomal RNA. II. Processing of mouse L-cell ribosomal RNA and variations in the processing pathway

Secondary structure mapping in the electron microscope was applied to ribosomal RNA and precusor ribosomal RNA molecules isolated from nucleoli and the cytoplasm of mouse L-cells. Highly reproducible loop patterns were observed in these molecules. The polarity of L-cell rRNA was determined by partial digestion with 3′-exonuclease. The 28 S region is located at the 5′-end of the 45 S rRNA precursor. Together with earlier experiments on labeling kinetics, these observations established a processing pathway for L-cell rRNA. The 45 S rRNA precursor is cleaved at the 3′-end of the 18 S RNA sequence to produce a 41 S molecule and a spacer-containing fragment (24 S RNA). The 41 S rRNA is cleaved forming mature 18 S rRNA and a 36 S molecule. The 36 S molecule is processed through a 32 S intermediate to the mature 28 S rRNA. This pathway is similar to that found in HeLa cells, except that in L-cells a 36 S molecule occurs in the major pathway and no 20 S precusor to 18 S RNA is found. The processing pathway and its intermediates in L-cells are analogous to those in Xenopus laevis, except for a considerable size difference in all rRNAs except 18 S rRNA.The arrangement of gene and transcribed spacer regions and of secondary structure loops, as well as the shape of the major loops were compared in L-cells, HeLa cell and Xenopus rRNA. The over-all arrangement of regions and loop patterns is very similar in the RNA from these three organisms. The shapes of loops in mature 28 S RNA are also highly conserved in evolution, but the shapes of loops in the transcribed spacer regions vary greatly. These observations suggest that the sequence complementarity that gives rise to this highly conserved secondary structure pattern may have some functional importance.     

Fine structure of ribosomal RNA. II. Distribution of methylated sequences within Xenopus laevis rRNA.

The distribution of methyl groups in rRNA from Xenopus laevis was analyzed by hybridization of rRNA to subfragments of either of two cloned rDNA fragments, X1r11 and X1r12, which together constitute a complete rDNA repeat unit. Using a mixture of 3H-methyl plus 32P-labelled rRNA as probe, the molar yield of methyl groups per rRNA region in hybrid could be calculated. For this calculation the length of the rRNA coding region in each DNA subfragment is needed, which was determined for X1r11 subfragments by the nuclease S1 mapping method of Berk and Sharp. The results show that both in 18S and 28S rRNA the methyl groups are nonrandomly distributed. For 18S rRNA, clustering was found within a 3' terminal fragment of 310 nucleotides. For 28S rRNA, clustering of methyl groups was found within a region of 750 nucleotides in length, which ends 500 nucleotides from the 3' end. In contrast, the 28S rRNA 5' terminal region of 900 nucleotides is clearly undermethylated. The general position of methyl groups in 28S rRNA correlates with the location of evolutionarily conserved sequences in this molecule, as recently determined in our laboratory.     

Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog Rana nigromaculata

The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions.     

Different forms of U15 snoRNA are encoded in the introns of the ribosomal protein S1 gene of Xenopus laevis.

Recent cloning and sequencing of one of the two Xenopus gene copies (S1b) coding for the ribosomal protein S1 has revealed that its introns III, V and VI carry a region of about 150 nt that shares an identity of 60%. We show here the presence in Xenopus oocytes and cultured cells of a 143-147 nt long RNA species encoded by these three repeated sequences on the same strand as the S1 mRNA and by at least one repeat present in the S1 a copy of the r-protein gene. We identify these RNAs as forms of the small nucleolar RNA U15 (U15 snoRNA) because of their sequence homology with an already described human U15 RNA encoded in the first intron of the human r-protein S3 gene, which is homologous to Xenopus S1. Comparison of the various Xenopus and human U15 RNA forms shows a very high conservation in some regions, but considerable divergence in others. In particular the most conserved sequences include two box C and two box D motifs, typical of most snoRNAs interacting with the nucleolar protein fibrillarin. Adjacent to the two D boxes there are two sequences, 9 and 10 nt in length, which are perfectly complementary to an evolutionary conserved sequence of the 28S rRNA. Modeling the possible secondary structure of Xenopus and human U15 RNAs reveals that, in spite of the noticeable sequence diversity, a high structural conservation in some cases may be maintained by compensatory mutations. We show also that the different Xenopus U15 RNA forms are expressed at comparable levels, localized in the nucleoli and produced by processing of the intronic sequences, as recently described for other snoRNAs.