Methylated bases and sugars in 16-S and 28-S RNA from L cells

332 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96180

METHYLATED BASES AND SUGARS IN 16-S AND 28-S RNA FROM L CELLS

B. G. LANE" AND TAIKI TAMAOKI Department o/ Biochemistry, and Cancer Research Unit (MeEaehern Laboratory), University o/ Alberta, Edmonton (Canada) (Received November ilth, 1968)

SUMMARY

I. Sugar-methylated and base-methylated compounds in alkali hydrolysates of L cell I6-S and 28-S RNA have been examined.

2. The extent of sugar methylation is much greater than the degree of base methylation in both I6-S and 28-S RNA.

3. The sugar-methylated nucleotides (OV-methyladenylate, OV-methylguany - late, 0V-methylcytidylate, OV-methyluridylate) are present in both I6-S and 28-S RNA.

4. Most of the sixteen possible alkali-stable sequences of the type NmpN, in which Nm is an 0V-methylated derivative of one of the four major nucleosides, have been recovered as dinucleoside phosphates from both I6-S and 28-S RNA.

5. The base-methylated nucleotides (NS-methyladenylate, NS-dimethyladeny - late) are present in I6-S RNA, and the base-methylated nucleotides (NS-methylade - nylate, 5-methylcytidylate) are present in 28-S RNA.

6. The analytical data have been evaluated and discussed in terms of the possibility that polynucleotides in I6-S RNA are heterogeneous, and also in terms of the possibility that I6-S polynucleotides may be involved in intracellular protein synthesis.

INTRODUCTION

There is limited methylation of between o.I and 6 °/o of the component bases, and of between o.I and 2 % of the component sugars in bulk tRNA and bulk rRNA from animal, plant, and microbial cells 1-13. Although net methylation of bases and sugars in bulk tRNA (2.5~7 °/o ) exceeds that in bulk rRNA (o.6-2 °/o), and base methylation in bulk tRNA (2-5 %) also exceeds that in bulk rRNA (O.l-O.5 °/o), it is often found that sugar methylation in bulk rRNA (o.1-2 ~o) is greater than that in bulk tRNA (o.3-1. 3 ~o)- Furthermore, whereas base methylation always exceeds the extent of sugar inethylation in bulk tRNA, sugar methylation often exceeds the degree of base methylation in bulk rRNA. For example, base methylation is three to four times more extensive than sugar methylation in bacterial rRNA 11,12, but the extent of sugar methylation far exceeds the degree of base methylation in plant rRNAI~,IS.

Abbreviations: NmpN stands for the OV-methylnucleosidyl-(3'-5')-phosphorylnucleoside with the N substituted for by the appropriate standard one-letter abbreviation for nucleosides.

* Present address: Department of Biochemistry, University of Toronto, Toronto, Ontaxio, Canada; to which address, communications in connection with this paper should be addressed.

Biochim. Biophys. Acta, 179 (1969) 332-34 °

M E T H Y L A T E D B A S E S A N D SUGARS IN RNA 333

Analyses for these base- and sugar-methylated components can provide a dis- cerning basis for distinguishing between bulk rRNA (or tRNA 9) specimens from different cells, on both quantitative and qualitative grounds, but they also afford an excellent means of chemically "fingerprinting" the slower- and faster sedimenting rRNA components from the same cell. For instance, the slower-sedimenting (I6-S) component of Escherichia coli rRNA contains N4,02'-dimethylcytidylate le and N ~- dimethyladenylate TM, both of which are not present in the faster-sedimenting (23-S) component. On the other hand, E. coli 23-S RNA contains 02'-methylguanylate,O 2'- methylcytidylate and 02'-methyluridylate 1~, all of which are virtually absent from E. coli I6-S RNA. It is the intention of this present report to record the results of a continuing investigation that was initiated in order to characterize, by analysis of trace components, the slower-sedimenting (I6-S) and the faster-sedimenting (28-S) rRNA components from an animal cell (L cell, mouse fibroblasts growing in tissue culture).

In an earlier report 1~, detailed evidence was presented to show that polynucleotides in I6-S RNA have a pattern of chain termini which is distinctively different from that found for polynucleotides in 28-S RNA, and preliminary evidence was presented to show that the pattern of sugar methylation in I6-S RNA is also quite different from that in 28-S RNA from L cells. In order to extend these preliminary observations on methylated components in L cell rRNA, we have undertaken to fully quantitate and characterize all of the base-methylated and sugar-methylated compounds present in alkali hydrolysates of the I6-S and 28-S components of L cell RNA. For this purpose, [Me-laCIRNA was prepared by aqueous phenol extraction

' of L cells that had been grown for one generation in a medium containing [Me-14C] - methionine. The EMe-14CJRNA was resolved into high and low molecular weight fractions by repeated precipitation from aqueous 2.5 M NaCI solution, at o °. The low molecular weight, NaCl-soluble polynucleotides (tRNA, 5-S rRNA, ADP-ribose polymer) were discarded, and high molecular weight, NaCl-insoluble polynucleotides were subjected to sucrose density gradient centrifugation in order to separate I6-S RNA from 28-S RNA. Each of the I6-S RNA and 28-S RNA specimens was hydrolyzed in alkali, and following dephosphorylation of the alkali hydrolysis products by treatment with E. coli alkaline phosphatase, the resulting base-methylated components were separated, as a group, from the sugar-methylated compounds, by one- dimensional filter-paper chromatography (see illustration in ref. 18). With the aid of authentic carrier compounds, individual base-methylated nucleosides, and individual sugar-methylated dinucleoside phosphates were isolated by two-dimensional paper-chromatographic resolution of the compounds in the two parent fractions. Indi- vidual methylated compounds were then quantitated by radioactivity measurements and further characterized by hydrolytic, chomatographic and electrophoretic techniques.

M A T E R I A L S A N D M E T H O D S

Preparation, hydrolysis and analysis o! [Me-14ClrRNA /rom L cells Detailed descriptions of the techniques used to prepare, hydrolyze and analyze

hydrolysates of I6-S and 28-S RNA from L cells have been presented in recent re- portslT, is.

Biochim. Biophys Acta, 179 (1969) 3 3 2 - 3 4 o

334 B. G. LANE, T. TAMAOKI

Characterization o/ methylated components derived ]rom L cell rRNA After resolving individual base-methylated nucleosides by two-dimensional fil-

ter-paper chromatography 19, each compound was eluted, a portion of the eluate was withdrawn for measurement of Me-14C radioactivity, and the remainder of the eluate was desalted by charcoal adsorption for further characterization of the nucleoside. Each base-methylated nucleoside was chromatographed in Solvents I (n-butanol- water-95 % ethanol (5 o:35:18, by vol.) (ref. 20)) and 2 (2-propanol-water-conc. HC1 (68:14. 4:17.6, by vol.) (ref. 2I)), and electrophoresed in Solvents 3 (I M formic acid, pH 2) and 4 (0.025 M tetraborate, pH 9.2), using a Durrum-type paper electrophoresis unit. Each chromatogram and electrophoretogram was sectioned into twenty or thir ty small areas, and each area was examined for 14C radioactivity and ultraviolet absorbance (of carrier nucleosides). In all cases, there was unambiguous coincidence of virtually all 14C radioactivity with the ultraviolet absorbance of the expected carrier nucleoside. Determination of complete radioactivity and absorbance distributions, between the origin and solvent front of every chromatogram and electrophoretogram, eliminates any dependence upon arbitrary reference standards such as RF values, which are notoriously dependent upon a host of (usually) uncontrolled variables ~2. Solvents I and 2 are useful for characterizing base-methylated nucleosides, since base methylation is associated with pronounced increments of chromatographic mobility, relative to unmethylated homologues 1,2. Electrophoresis in Solvent 3 is useful for establishing that a particular base-methylated nucleoside has the same electrical charge properties as its unmethylated homologue at pH 2 (ref.I6). Electro- phoresis in Solvent 4 is useful for establishing whether methylation is in the base, or in the sugar, of a given nucleoside 16.

After resolving individual sugar-methylated dinucleoside phosphates by two- dimensional chromatography 6,16, each dinucleoside phosphate (NmpN) was eluted, a portion of the eluate was withdrawn for measurement of ~4C radioactivity, and the remainder of the eluate was desalted by charcoal adsorption for further characterization. Each of the dinucleoside phosphates was subjected to electrophoresis in Sol- vent 3. Every electrophoretogram was sectioned into several small areas, and each area was examined for ~4C radioactivity and ultraviolet absorbance. In all cases, there was unambiguous correspondence between 14C radioactivity and the absorbance of the expected carrier dinucleoside phosphate, although it is perhaps worth noting that some isomeric pairs, particularly AmpG-GmpA, were partially resolved, a re- sult that is not unexpected from earlier studies with unmethylated dinucleoside phosphates (see ref. 23). Electrophoresis in Solvent 3 is useful since the characteristic relative mobilities for different NmpN compounds are predictable from the mobilities of their component nucleosides, at pH 2. Certain pairs of isomeric dinucleoside phosphates (particularly AmpG-GmpA and CmpU-UmpC, but to a lesser degree, GmpC-CmpG, and GmpU-UmpG) are not well-resolved during the primary resolution of NmpN compounds by two-dimensional paper chromatography (see ref. 16, for illustration). However, the proportion of each isomer in a mixture was readily determined in the course of the hydrolytic degradations used to characterize the dinucleoside phosphates. Each NmpN compound was hydrolyzed by treatment with the enzymes in whole snake venom ~, which release the component phosphate, nor- mal nucleoside and OZ'-methylnucleoside, in equimolar proportions. The radioactive component (the sugar-methylated nucleoside) released by venom treatment was sub-


METHYLATED BASES AND SUGARS II~ RNA 335

jected to electrophoretic characterization in Solvents 3 and 4, and to chromatographic characterization in Solvent 5 (76 % aqueous ethanol developing solvent, used with ammonium sulphate-impregnated paper (ref. 24)). The sugar-methylated nucleosides display pronounced increments in mobility, relative to the unmethylated homologues in Solvent 5 (see ref. I5), and the usefulness of the electrophoretic solvents has been indicated earlier.

RESULTS

The data presented in Table I summarize the results of analyses for base-methylated nucleosides and sugar-methylated dinucleoside phosphates (NmpN), which were prepared by enzymic removal of monoester phosphate from the products formed when I6-S and 28-S L cell RNA were separately hydrolyzed in alkali. The amount of radioactivity incorporated into the carbon skeletons of each of the four major ribo- nucleosides has also been entered in Table I for the record. The methylated components (i.e., base-methylated nucleosides and sugar-methylated dinucleoside phosphates) account for about 2 % of the total constituent nucleosides in L cell rRNA, and as a consequence, they contain about 2 % of the total radioactivity that is incorporated into carbon skeletons. Since methylated components contain about 5 ° % of the total 14C radioactivity in the RNA specimens, but only about 2 % of the total 14C radioac- t ivity is in their carbon skeletons, it can be concluded that about 98 % of the 14C radioactivity found in methylated components is confined to their methyl substituents. Correction for a small amount of carbon skeleton labeling in the methylated components has been deemed negligible, and no such correction has been entered for the figures in Table I.

The amount of Me-14C radioactivity, per mole of constituent nucleoside in I6-S RNA is about 1.3 times greater than in 28-S RNA (i.e., 80 168/61 3Ol). Since the half-life for the generation of I6-S RNA is about IO % shorter than for 28-S RNA in L cells ~5, the degree of methylation in I6-S RNA is probably only about 1.2 times greater than in 28-S RNA. Furthermore, since OV-methylribose comprises 1.o 5 % of the total sugars in bulk L cell rRNA 2s, and since sugar methylation accounts for about 9 ° % of the net methylation in L cell rRNA (Table I), it can be concluded that there is methyl substitution of roughly 1.3 % of the nucleosides in I6-S RNA, and of roughly I . I % of the nucleosides in 28-S RNA.

Since the results in Table I were obtained after alkali hydrolysis of the RNA preparations, it is pertinent to note that alkaline conditions induce conversion of I-methyladenosine to N6-methyladenosine, of 7-methylguanosine to 2,6-diamino- 4- hydroxy-5-methylformamidopyrimidine, and of 3-methylcytidine to 3-methyluridine 27-29. Since I-methyladenosine, 7-methylguanosine and 3-methylcytidine (as well as 3-methyluridine) are known to occur naturally in some types of RNA it is possible that in the present study, Ne-methyladenosine was derived from i-methyladenosine in the course of the alkali-catalyzed hydrolysis of RNA, and that the alkali-conversion products of 7-methylguanosine and 3-methylcytidine were not detected. Be- cause 3-methylcytidine and 3-methyluridine were not found in the rRNA from another animal source 3°, and since 7-methylguanosine is present in only a very small quanti ty


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METHYLATED BASES AND SUGARS IN R N A 337

in bacterial rRNA 14, and not at all in yeast rRNA 14, it seems reasonable to conclude that the amounts of any undetected base-methylated nucleosides would not signif- icantly alter the general conclusion that the degree of sugar-methylation is vastly greater than the degree of base-methylation in the rRNA from this cell of animal origin.

Notably, N6-dimethyladenylate is virtually confined to I6-S RNA and 5-methylcytidylate is virtually confined to 28-S RNA. On the other hand, N6-monomethyl - adenylate, OV-methyladenylate, OV-methylguanylate, 0V-methylcytidylate and 03'- methyluridylate are found in both I6-S and 28-S RNA. All of the different NmpN sequences, with the possible exception of UmpU, are present in both I6-S and 28-S RNA, also. Most, and possibly all, of the UmpU found in 28-S RNA may arise by (i) de-amination of CmpU, UmpC and CmpC during alkali-catalyzed hydrolysis of the RNA, and (ii) limited contamination of 28-S RNA by I6-S RNA.

DISCUSSION

The polynucleotides in L cell I6-S RNA have a mean residue length of about 14oo nucleotides 17. As a consequence of this, a particular nucleotide that is present once in every ribonucleate chain will account for o.075 ~o of the total nucleotides in L cell I6-S RNA. Since OV-methylated nucleotides account for 1.2 % of the total nucleotides in L cell I6-S RNA, there is an average of sixteen OV-methylated dinucleotide sequences per polynucleotide chain. Furthermore, since there are sixteen different OV-methylated dinucleotide sequences in L cell I6-S RNA (Table I), each of these different sequences would account for one-sixteenth (6.25 %) of the total NmpN sequences, if all were present in every polynucleotide chain. From the data in Table I, it is apparent that some of the sequences are not present in all of the polynucleotides in L cell I6-S RNA. For instance, the nucleotide OV-methyladenylate, in the sequence AmpA, accounts for o.13-1.2 = o.16 % of the total nucleotides, and could occur twice in every polynucleotide chain, but 0V-methylcytidylate, in the sequence CmpU, accounts for 0.03" 1.2 = 0.o36 % of the total nucleotides and could not be present in more than about one-half of the polynucleotides in L cell I6-S RNA. The evidence that some sugar-methylated sequences are present in only a fraction of the total polynucleotides in L cell I6-S RNA can be extended to include the base- methylated nucleotides, N6-methyladenylate and N6-dimethyladenylate. For example, N6-dimethyladenylate with its two methyl substituents, contains only one-sixth as much radioactivity as the monomethylated sequence AmpA (Table I). Consequently, N6-dimethyladenylate accounts for o.13" 1.2/12 = o.o13 % of the total nucleotides, and could be present in only about one-sixth of the polynucleotides in the L cell I6-S RNA preparation that was examined in the present investigation. These foregoing observations serve to supplement an ever-expanding body of chemical, physical, cytological and biological evidence which indicates that rRNA may be more heterogeneous than is generally recognized to be the case:

(i) Chemical evidence o[ heterogeneity in rRNA. Analyses for major nucleotides show that polynucleotides in the slower- and faster-sedimenting components of rRNA are chemically distinguishable, and analyses for minor components show that there


338 B.G. LANE, T. TAMAOKI

are different polynucleotides within each of the slower- and faster-sedimenting components. When ribonucleates are isolated by aqueous phenol extraction of wheat embryo, and then repeatedly precipitated from aqueous I M NaC1 in order to remove low molecular weight polynucleotides (tRNA, 5-S rRNA, ADP-ribose polymer), it is found that the RNA preparations contain only two, sharply sedimenting (I8-S and 28-S) components when they are examined in the analytical ultracentrifuge 6. How- ever, these bulk rRNA preparations from a plant organism contain more than two polynucleotide species, since roughly equal amounts of the four major ribonucleo- sides are found at both 3'-hydroxyl and 5'-phosphomonoester termini 15. Terminal heterogeneity has also been demonstrated for polynucleotides in both the slower- and faster-sedimenting rRNA components from microbial 31-83 and animal ~ cells, including L cells 17.

(ii) Physical evidence o[ heterogeneity in rRNA. Electrophoretic analyses of fractions obtained by sucrose density-gradient sedimentation have established the pres- ence of many more than two polynucleotide species in bulk rRNA from plant organisms and organs 35, and in bulk rRNA from animal organs and cells 35-3~. In particular, extensive electrophoretic heterogeneity has been observed for the fraction of polynucleotides that sediments between the two principal sedimentational components of bulk rRNA from animal organs ~¢. These latter observations have special relevance to this present study of L cell RNA, in which sucrose density-gradient fractions were arbitrarily selected in order to minimize cross-contamination in the "purified" I6-S and 28-S RNA specimens. Thus, a fraction of I6-S polynucleotides, enriched in N~-dimethyladenylate, could have been preferentially lost during preparation of the I6-S RNA specimen examined in this present investigation. This possibility was first suggested to us by the observation that the total amount of N6-di - methyladenylate in the density-gradient-purified I6-S and 28-S RNA specimens was appreciably lower than had been expected from the results of our earlier analyses of bulk rRNA 18 which had not been subjected to purification by density-gradient centrifugation. This possibility has been given further substance by the observations contained in a recent report by Zimmerman 38, who recovered N6-dimethyladenylate from HeLa cell I6-S RNA in an amount that was only one-half as great as would have been expected if all of the polynucleotides in HeLa cell I6-S RNA contain N~-di - methyladenylate. Notably, Zimmerman's data show that N~-dimethyladenylate is concentrated at the leading edge of the I6-S peak during sucrose density-gradient centrifugation of HeLa cell rRNA, and it is this fraction of polynucleotides that was preferentially lost in preparing L cell I6-S RNA that was used for the present study.

(iii) Cytological evidence o[ heterogeneity in rRNA. Although it is not now possible to assess either the extent, or weight distribution, of heterogeneity within bulk rRNA from cells, organs and organisms, there is an obvious subcellular basis that can be expected to contribute to such heterogeneity. For example, different sedimentation properties and different degrees of methylation are observed when mito- chondrial rRNA is compared with rRNA from the soluble cytoplasm of the same animal cell 39. In the case of plant cells, it is observed that the sedimentation 4° and electrophoretic ~5 properties of chloroplast rRNA differ from the corresponding properties of rRNA isolated from the soluble cytoplasm of the same cell.

(iv) Biological evidence o] heterogeneity in rRNA. Another relevant basis for heterogeneity in the polynucleotides that comprise cellular I6-S RNA can be antic-

Biochim. Biophys. Acta, 179 (t969) 332-34 o

METHYLATED BASES AND SUGARS IN R N A 339

ipated from a growing body of evidence which indicates that " template" RNA from mammalian cells appears to have a mean sedimentation coefficient of about I6-S 41. Whether or not "template" RNA derives from an extraribosomal component of a polysomal complex 4~, or is an integral component of the smaller ribosomal subunit 48m, this type of RNA would be expected to be recovered partially, or completely, in the bulk I6-S RNA isolated from L cells by the techniques employed in our investiga- tions. HARRIS has recently suggested cogent reasons for thinking that the "templates" for intracellular protein synthesis, in animal cells, can be expected to be found in the cellular I6-S RNA ~. It is clear that if I6-S RNA contains the "templates" for intracellular protein synthesis, there could be extensive heterogeneity in the bulk I6-S RNA from animal cells. The most immediate, and unresolved problem, would appear to be whether or not such "templates" comprise a major, or minor fraction, of the polynucleotides in cellular I6-S RNA. Possibly the bulk of the I6-S RNA, from the smaller ribosomal subunits, is composed of the "long-lived templates" which have been invoked to account for translational (cytoplasmic) control of protein synthesis, whereas a relatively small fraction of I6-S RNA may be composed of the "short-lived templates" that have been invoked to account for transcriptional (genomic) control of protein synthesis, and which have been detected as nascent I6-S RNA in short-term labeling experiments 4~.

The fact that the bulk I6-S RNA has a characteristic pattern of OV-methylated dinucleotide sequences, and the fact that there is substantial similarity between the OV-methylation patterns of L cell I6-S RNA and HeLa cell I6-S RNA 45, would seem to have a significant bearing on any proposed or proven involvement of I6-S RNA in cellular metabolism. For instance, if most of the polynucleotides in I6-S RNA are, in fact, "templates" for intracellular protein synthesis (e.g. ribosomal proteins, in the case of bacteria46), then there is, apparently, either a fairly uniform collection of the same "templates" in distantly related cells (L cell and HeLa cell), or a remarkable homology among the different templates from different cell types, with respect to their overall pattern of OV-methylation.

Methylation of bacterial rRNA, being preponderantly directed toward bases rather than sugars, may have an effect which is different from the influence of rRNA methylation in animal systems, where methylation is preponderantly directed toward sugars, to yield a Io-fold greater proportion of OV-methylribose in animal rRNA than is found in bacterial rRNA. Nonetheless, it should be mentioned in the present con- text, that "undermethylated" bacterial rRNA is a more efficient "template" for protein synthesis in vitro than is the corresponding "fully methylated" bacterial rRNA 4e. However, since the template efficiency of fully methylated bacterial rRNA in vitro can be markedly enhanced (some 20- or 3o-fold) by the addition of neomycin 47, it is not possible, at present, to relate the results of experiments performed with in vitro systems to a situation, in vivo, or at least intracellularly, where the engagement of "templates" in a protein-synthesizing complex will undoubtedly involve conditions that are not (ordinarily) fulfilled in vitro.

Finally, it should be noted that the pattern of OV-methylation in L cell 28-S RNA shows substantial homology with that reported for HeLa cell 28-S RNA 45. Until uncertainties surrounding the measurements of mean molecular weight for L cell 28-S RNA 1~ and HeLa cell 28-S RNA 48 have been satisfactorily resolved, any discussion of quantitative data for methylated components would be premature.

Biochim. Biophys. Acta, 179 (1969) 332-34 o

34 ° B. G. LANE, T. TAMAOKI

ACKNOWLEDGMENTS

We are grateful to Mrs. Carol J. Tilley for her skillful technical assistance in this investigation. We also wish to express gratitude to the Medical Research Council of Canada for continuing financial support of these studies (MRC-MA-I226 and MRC-MA-I953 ).

R E F E R E N C E S

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