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.     

Demonstration of the "5-8 S" ribosomal sequence in HeLa cell ribosomal precursor RNA

HeLa cell “5.8 S” ribosomal RNA was digested with T₁ ribonuclease and the digestion products were characterized. In particular several hexa-, or larger, oligonucleotides were well fractionated by T₁ ribonuclease plus alkaline phosphatase fingerprints. The sequences of these large products were determined. The same large products were identified in fingerprints of “native” 28 S RNA, that is, 28 S RNA to which 5.8 S RNA is attached. The products were demonstrably absent in fingerprints of heat-denatured 28 S RNA, which lacks the 5.8 S fragment. The oligonucleotides were present in fingerprints of 32 S RNA, whether previously heated or not. One of the largest 5.8 S oligonucleotides contains an alkali-stable (2′-O-methylated) dinucleotide, Gm-C. This product was identified in fingerprints of methyl-labelled 45 S RNA. These findings prove that the 5.8 S ribosomal sequence is present within HeLa cell ribosomal precursor RNA. In addition to the methylated nucleotide, two pseudouridylate residues were discovered in HeLa cell 5.8 S RNA.     

2'-O-methylated oligonucleotides in ribosomal 18S and 28S RNA of a mouse hepatoma, MH 134.

Simple two-dimensional thin-layer chromatography was found to be useful for the separation of sugar methylated dinucleotides in RNA. Of the 16 possible sequences of the type Nm-Np, 15 were separated and all the sequences were determined. In a mouse hepatoma, MH 134, the levels of the sugar methylation in the 18S and 28S RNA molecules were 17-18 and 11-12 per 1000 nucleotides, respectively. Thus, 18s RNA contained approximately 35 2'-O-methylated dinucleotides and 28S RNA approximately 60 2'-O-methylated dinucleotides. The pattern of distribution was also distinct between these two molecules. Two 2'-O-methylated trinucleotides were identified in the 28S RNA with the sequences Um-Gm-Up and Um-Gm-psip. A unique 2'-O-methylated tetranucleotide was present also in the 28S RNA, the sequence of which was Am-Gm-Cm-Ap. The 5'-terminal nucleotides of both 18S and 28S RNA were obtained as nucleoside 3',5'-diphosphates (pNp) in the trinucleotide fraction of the RNase T2 digest. The 5'-termimi of 18S and 28S RNA were pUp and pCp, respectively, and found to be almost homogeneous.     

Nucleotide sequence neighbouring a late modified guanylic residue within the 28S ribosomal RNA of several eukaryotic cells.

The nucleotide sequence of a particular T1 oligonucleotide found in 41S and 28S RNAs of several cellular cell lines (human, mouse, rat and chicken fibroblast) but absent in 45S ribosomal RNA has been deduced. Its primary structure : A-U-U*-G*-psi-U-C-A-C-C-C-A-C-U-A-A-U-A-Gp shows the presence of a modified G residue which explains the existence of this oligonucleotide in the T1 fingerprint of 41S RNA and 28S. Its absence on the 45S RNA T1 fingerprint is accounted for by a late modification.     

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.     

Secondary structure maps of ribosomal RNA and DNA: I. Processing of Xenopus laevis ribosomal RNA and structure of single-stranded ribosomal DNA

Precursor and mature ribosomal RNA molecules from Xenopus laevis were examined by electron microscopy. A reproducible arrangement of hairpin loops was observed in these molecules. Maps based on this secondary structure were used to determine the arrangement of sequences in precursor RNA molecules and to identify the position of mature rRNAs within the precursors. A processing scheme was derived in which the 40 S rRNA is cleaved to 38 S RNA, which then yields 34 S plus 18 S RNA. The 34 S RNA is processed to 30 S, and finally to 28 S rRNA. The pathway is analogous to that of L-cell rRNA but differs from HeLa rRNA in that no 20 S rRNA intermediate was found. X. laevis 40 S rRNA (M_r = 2.7 × 10⁶) is much smaller than HeLa or L-cell 45 8 rRNA (M_r = 4.7 × 10⁶), but the arrangement of mature rRNA sequences in all precursors is very similar. Experiments with ascites cell 3′-exonuclease show that the 28 S region is located at or close to the 5′-end of the 40 S rRNA.Secondary structure maps were obtained also for single-stranded molecules of ribosomal DNA. The region in the DNA coding for the 40 S rRNA could be identified by its regular structure, which closely resembles that of the RNA. Regions corresponding to the 40 S RNA gene alternate with non-transcribed spacer regions along strands of rDNA. The latter have a large amount of irregular secondary structure and vary in length between different repeating units. A detailed map of the rDNA repeating unit was derived from these experiments.Optical melting studies are presented, showing that rRNAs with a high (G + C) content exhibit significant hypochromicity in the formamide/urea-containing solution that was used for spreading.     

Primary structure of rabbit 18S ribosomal RNA determined by direct RNA sequence analysis

The primary structure of rabbit 18S ribosomal RNA was determined by nucleotide sequence analysis of the RNA directly. The rabbit rRNA was specifically cleaved with T1 ribonuclease, as well as with E. coli RNase H using a Pst 1 DNA linker to generate a specific set of overlapping fragments spanning the entire length of the molecule. Both intact and fragmented 18S rRNA were end-labeled with [32P], base-specifically cleaved enzymatically and chemically and nucleotide sequences determined from long polyacrylamide sequencing gels run in formamide. This approach permitted the detection of both cistron heterogeneities and modified bases. Specific nucleotide sequences within E. coli 16S rRNA previously implicated in polyribosome function, tRNA binding, and subunit association are also conserved within the rabbit 18S rRNA. This conservation suggests the likelihood that these regions have similar functions within the eukaryotic 40S subunit.     

Complementarity between ferritin H mRNA and 28 S ribosomal RNA

We have found an interesting complementarity in sequences of human ferritin H mRNA and 28 S ribosomal RNA. Immediately upstream of the initiating AUG in the ferritin mRNA is a stretch of 67 nucleotides which contains sequences complementary to several regions in 28 S RNA. One such region can form 55 base pairings with the 5' noncoding region of the ferritin H mRNA. Most of the complementarity is due to repeats of CCG in the ferritin mRNA and GGC in the ribosomal RNA. The regions of complementarity in the 28 S RNA appear to be expansion sequences that have arisen in the evolution of eukaryotic ribosomal RNA. We suggest that interaction of ferritin mRNA and 28 S RNA may function to regulate the stability and/or translatability of ferritin mRNA.     

3'-terminal processing of ribosomal RNA precursors in mammalian cells.

The 3'-terminal structures of ribosomal 28S RNA and its precursors from rat and mouse were analyzed by means of periodate oxidation followed by reduction with 3H-borohydride. 3'-terminal labeled nucleoside derivatives produced by RNase T2 digestion were determined by thin-layer chromatography and oligonucleotides generated by RNase T1 digestion were analyzed by DEAE-Sephadex chromatography. In the rat, the major 3'-terminal sequences of ribosomal 28S RNA, nucleolar 28S, 32S, 41S, and 45S RNAs were YGUoh, GZ2Uoh, GZ12Uoh, GZ2Uoh, and GZ7Goh, respectively, whereas in the mouse corresponding sequences were YGUoh, GZ1,2, or 3Uoh, Goh, Uoh and GZ 13Uoh, respectively. (Y: pyrimidine nucleoside, Z: any nucleoside other than guanosine) These results suggest that a "transcribed spacer" sequence is present at the 3'-terminus of the 45S pre-ribosomal RNA, which is gradually removed during the steps of processing.     

Heterogeneity in Chinese hamster ribosomal DNA

A discrete heterogeneity has been detected in Chinese hamster ribosomal DNA after Eco R1 digestion of total DNA followed by a Southern transfer and hybridization with [¹²⁵I]18S or [¹²⁵I]28S ribosomal RNA. Digestion with Eco R1 produces three fragments, 4.3, 6.0 and 9.5×10⁶ daltons respectively, which hybridize with 18S RNA. The smallest fragment also hybridizes with 28S RNA. Either length heterogeneity or sequence heterogeneity (i.e. presence of an additional Eco R1 site in some of the rDNA molecules) must be invoked to account for the two larger Eco R1 fragments that contain 18S but not 28S sequences. Eco R1 and Hind III maps, consistent with either length or sequence heterogeneity, are presented. The data at this time, however, do not distinguish between the two alternatives.     

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.     

Synthesis and maturation of ribosomal ribonucleic acids in isolated HeLa cell nuclei. A tracer study on the topology of the 45S precursor of ribosomal ribonucleic acids

The synthesis and processing of RNA by isolated HeLa cell nuclei was studied at low ionic strength in the presence of alpha-amanitin. The RNA polymerase reaction, with endogenous template and enzyme, rapidly reaches a plateau dependent on the amount of nuclei. Evidence is presented that incorporation of [(3)H]UMP proceeds only in growing RNA chains, whereas initiation of new RNA chains is arrested. The product formed contains all the main components of the 45S pre-rRNA (precursor of rRNA) maturation pathway (45S, 32S and 20S pre-rRNA; 28S and 18S rRNA). Most of the labelled material is in the mature rRNA components and their immediate precursors, even at very short times of incubation (2min). Small, but definite, 5S and 4S RNA peaks are also observed. At shorter incubation times a substantial amount of [(3)H]UMP is incorporated into RNA molecules in the 24S and 10-16S zones. This RNA material is considered to represent the non-conserved segments of 45S pre-rRNA in the process of nucleolytic degradation. A model for the tracer study of the topology of 45S pre-rRNA, on arrest of rRNA initiation, is discussed. The experimental evidence obtained supports the following structure of 45S pre-rRNA: 5'-end-28S rRNA unit-18S rRNA unit-nonconserved segment-3'-end.     

Processing of ribosomal RNA in a temperature sensitive mutant of BHK cells.

The processing of ribosomal RNA has been studied in a temperature sensitive mutant of the Syrian hamster cell line BHK 21. At 39 degrees C, these cells are unable to synthesize 28S RNA, and 60S ribosomal subunits, while 18S RNA, and 40S subunits are produced at both temperatures. At 39 degrees C the 45S RNA precursor is transcribed and processed as in wild type cells. The processing of the RNA precursors becomes defective after the cleavage of the 41S RNA, and the separation of the 18S and 28S RNAs sequences in two different RNA molecules. The 36S RNA precursor, which is always present in very small quantity in the nucleoli of wild type cells and of the mutant at 33 degrees C, is found in very large amounts in the mutant at 39 degrees C. The 36S RNA can be, however, slowly processed to 32S RNA. The 32S RNA cannot be processed at 39 degrees C, and it is degraded soon after its formation. Only a small proportion accumulates in the nucleoli. The 32S RNA synthesized at 39 degrees C cannot be processed to 28S RNA upon shift to the permissive temperature, even when the processing of the newly synthesized rRNA has returned to normal. The data suggest that the 36S and 32S RNAs are contained in aberrant ribonucleoprotein particles, leading to a defective processing of the particles as a whole.     

Chromosome location of the ribosomal genes in Triturus vulgaris meridionalis (Amphibia Urodela)

In Triturus vulgaris meridionalis, the 18S + 28S rDNA sequences have been shown to be located in a number of additional chromosomal sites besides the nucleolus organizing region. The additional ribosomal sites have been found to vary as to their number and chromosomal location in different individuals of the species.—The data presented in this study concern the chromosomal distribution of the ribosomal sequences as analyzed by in situ hybridization technique in two individuals as well as in their offspring. The evidence obtained by this analysis indicates quite clearly that all 18S + 28S rRNA sites present in each individual genome are inherited according to simple mendelian principles.Abbreviations rRNA ribosomal RNA - NOR nucleolus organizer region - rDNA DNA coding for 18S+28S rRNA plus the intervening spacers - SSC 0.15M Sodium chloride, 0.015 M Sodium citrate, pH 7 - RNase ribonuclease     

Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids.

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6X10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5'-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8X10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25X10(6) mol.wt.(41S), a precursor common to both mature rRNA species ; 2.60X10(6)(36S) and 2.15X10(6)(32S) precursors to 28S rRNA; 1.05X10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S leads to 41S leads to 32S+21S leads to 28S+18S rRNA and (ii) 45S leads to 41S leads to 36S+18S leads to 32S leads to 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9X10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.     

Effect of point mutations on 5.8S ribosomal ribonucleic acid secondary structure and the 5.8S--28S ribosomal ribonucleic acid junction

Naturally occurring differences in the nucleotide sequences of 5.8S ribosomal ribonucleic acids (rRNAs) from a variety of organisms have been used to study the role of specific nucleotides in the secondary structure and intermolecular interactions of this RNA. Significant differences in the electrophoretic mobilities of free 5.8S RNAs and the thermal stabilities of 5.8S--28S rRNA complexes were observed even in such closely related sequences as those of man, rat, turtle, and chicken. A single base transition from a guanylic acid residue in position 2 in mammalian 5.8S rRNA to an adenylic acid residue in turtle and chicken 5.8S rRNA results both in a more open molecular conformation and in a 5.8S--28S rRNA junction which is 3.5 degrees C more stable to thermal denaturation. Other changes such as the deletion of single nucleotides from either the 5' or the 3' terminals have no detectable effect on these features. The results support secondary structure models for free 5.8S rRNA in which the termini interact to various degrees and 5.8S--28S rRNA junctions in which both termini of the 5.8S molecule interact with the cognate high molecular weight RNA component.