THE DE'fERMiNATEON OF EkQ-ME‘E‘HYLATION {‘3 RNA Thesis for {The {Degree 0‘; pk. D. HECEEME‘? S'E'ETE UNE‘JERSE?Y Joseph S. Abba’ae 297': rH'f ---- LIBRARY Michigan State ; University Joseph S. Abbate ABSTRACT A technique was developed for the determination of 2'jg- methylation in RNA using gas chromatography to quantitate the methanol released by perchloric acid cleavage of the 2'-methoxy group. The technique could be used with small samples of RNA and did not require radioactive labeling. Base methylated com- pounds were shown not to interfere with the assay and the method was found to be accurate over widely ranging contents of 2'-Qfimethy1nucleotides. This technique was used to measure levels of 2'-Qfmethylation in the rRNA and tRNA of various organisms. Seven species of procaryotes and twelve eucaryotes ranging from yeast to several mamalian species were included in the survey. Bacterial ribo- somal RNAs were found to have sugar methyl contents of from 0.2 to 0.6% while tRNA of the thermophile Thermus aquaticus was methylated to the extent of 1.2%. The rRNA of higher organ- isms had a range of 1.1 to 1.7% 2'-Qfmethylation. Thus, 2'19- methylation of rRNA occurs only over a narrow range, although real differences between Species exist. The 2'-Qfmethy1 levels in rRNA were shown to remain con- stant in early development in Xenopus laevis from the ovarian egg stage to the time of hatching. At this latter stage, some 50% of the original ovarian egg rRNA has been replaced by newly synthesized rRNA. Likewise, in Musca domestica, the sugar methyl- ation levels remain the same throughout the life history of this insect although extensive degradation of the larval tissues Joseph S. Abbate takes place during pupation to be followed by generation of the adult insect from the imaginal discs. An investigation of tumor cells as compared with their normal counterparts showed that the 2'-Qfmethy1 contents of the rRNAs were similar but that in all cases examined, the tumor total nuclear RNA was more highly 2'-Qfmethy1ated than was normal nuclear RNA. This result is discussed in light of theories of information transfer from the nucleus to the cyto- plasm. The small molecular weight nuclear RNA species were shown to have as a group the highest sugar methyl content measured for any RNA, 2.2% in Novikoff hepatoma cells; 1.9% in rat liver. The data are consistent with 2'-Qfmethylation of ribosomal RNA being a Species specific constant value which is not affected by the rate of RNA synthesis or gross physiological change in the organism. THE DETERMINATION OF 2'-97METHYLATION IN RNA By Joseph S. Abbate A THESIS Submitted to ‘Michigan State university in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemisty 1971 G\ \_3 ta “5* Q‘ ey- Dedicated to my father and mother. ii ACKNOWLEDGMENTS First, I would like to thank Dr. Fritz Rottman for his guidance and help in all phases of this project. In addition, because of the wide scope of the experiments to be described, thanks are also due to a number of people: Dr. J. Shaver and his laboratory provided instruction and assistance in techniques of frog handling and experimentation. Dr. W. W. wells was of great help in setting up the gas chromato- graph. Also, Dr. L. Bieber initiated us in the mysteries of the house fly and was very generous in his donation of materials and time. In my own laboratory, I would particularly like to thank Galvin Swift for her fine initial work on 2'-Qfmethylation of y. domestica RNA and for her help in later preparations of RNA from these creatures. Brian Dunlap provided many of the bac- terial RNAs used in this work as well as sharing the trials of RNA extraction from gels. Karen Friderici also prepared several of the RNA species and in addition provided much technical assistance (i.e., cutting out peaks from the GC tracings). I must also thank Lee Pike for the use of his wheat germ RNA and gas chromatograph and Diana Filner for A, salina RNA. I would also like to thank Ronald Desrosiers, so he won't feel bad. iii TYPIST'S ACKNOWLEDGMENT For all her dedication and hours of work on this thesis I would like to give my profound thanks to the former Joyce Hallett. She hasn't been bad as a wife either. iv TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS LITERATURE REVIEW Introduction Historical 2'-97Methy1 Levels in Ribosomal and Transfer RNA Other RNA Species Distribution of 2'-97Methy1ation Problematical Cases Low Molecular Weight Nuclear RNA Ribosomal RNA Processing (Eucaryotic Organisms) Ribosomal RNA Processing (Procaryotic Organisms) The Effect of 2'-QfMethylation on Enzymes of RNA Metabolism Synthetic 2'-QfMethyl Compounds Speculations on Function METHODS Determination of 2'-97Methylation by Gas Chromato- sraphy Abbate, J. and F. Rottman (1972). A Gas Chromato- graphic Method for the Determination of 2'1Q-Methyl- ation in RNA. Anal. Biochem., in_press. Preparation of RNA Xenopus laevis : Collection of Materials vii viii ix 10 13 15 19 21 21 25 25 26 so 52 Xenopus laevis : Preparation of RNA Musca domestica : Collection of Materials Musca domestica : Preparation of RNA CytOplasmic and Nuclear RNA from Normal and Tumor Cells Determination of 2'jQ-Methylation by Column Chromato- graphy Analysis of Dinucleotides Density Gradient Electrophoresis Preparative Sucrose Gradients Polyacrylamide Gel Electrophoresis RESULTS Introduction Survey of 2'-97Methylribose Content in RNA Studies with Amphibia Studies with Diptera Studies with Tumor Cells Introduction RNA Separation Comparison of 2'-97Methy1ation in Normal and Tumor Cells DISCUSSION REFERENCES vi 53 53 5h 55 56 57 59 59 6O 61 62 66 72 79 79 85 89 96 II III IV VI VII VIII IX XI XII XIII XIV LIST OF TABLES 2'-QfMethylation in Ribosomal and Transfer RNA 2'-97Methylation of Ribosomal RNA Components Distribution of 2'-QfMethylation among the Constit- uent Nucleosides 2'-QfMethylation and rRNA Processing in Novikoff Hepatoma Cells 2'-97Methylation in Procaryotic Organisms 2'-QfMethylation in Eucaryotic Organisms DEAE-Cellulose Column Chromatography 2'-QfMethylation in Xenopus laevis by Column Chrom- atography 2'-O-Methylation of Xenopus laevis RNA 2'-QfMethylation of Musca domestica Total RNA 2'-97Methylation of Musca domestica rRNA 2'-97Methylation of Drosophila melanogaster RNA Extraction of RNA from Polyacrylamide Gels 2'-97Methylation in Normal and Tumor Cells Fractionated RNA from Rat Liver and Novikoff Cells vii ll 16 65 65 68 70 71 75 75 78 81 8h 86 LIST OF FIGURES Processing of MES Nucleolar RNA Penman, 1970) viii (Weinberg and 18 260 AMP, ADP dAMP CAM Cm CMP, CDP CTAB DEAE DNA EDTA GC GMP, GDP mA mRNA N4MeCmp Poly (A), Poly (C), Poly (G), Poly (I), Poly (U) Poly (Am, C) Poly (Cm, U) LIST OF ABBREVIATIONS Absorbancy at 260 nm 2'-97Methy1adenosine Adenosine mono-, diphOSphate Deoxyadenylic acid Chloramphenicol 2'-Q:Methylcytosine Cytidine mono-, diphosphate Cetyltrimethylammonium bromide Diethylaminoethyl Deoxyribonucleic acid Ethylenediamine tetra-acetic acid Gas chromatography 2'-97Methy1guanosine Guanosine mono—, diphOSphate Milliamperes Messenger RNA N4-Methyl-2'~97methy1cytidy1ic acid Homopolymers of adenylic acid, cytidylic acid, guanylic acid, inosinic acid and uridylic acid Random heteropolymer of 2'-Qfimethyladeny1ic and cytidylic acid Random heteropolymer of 2'-Qfmethy1cytidy1ic acid and uridylic acid ix PV rR SD n 5 TE Tr tR Um UM PVS rRNA SDS snRNA TEAB Tris tRNA Um UMP, UDP 28S A RNA Polyvinylsulfate Ribonucleic acid Ribosomal RNA Standard deviation Sodium dodecyl sulfate Small molecular weight nuclear RNA Triethylammonium bicarbonate Tris(hydroxymethyl)aminomethane Transfer RNA 2'-Qfmethyluridine Uridine mono-, diphosphate Ultraviolet 28$ Associated RNA moiet all f found germ 1 PM me LITERATURE REVIEW Introduction Ribonucleic acid is methylated in both the base and sugar moieties (Starr and Sells, 1969). Base methylation occurs on all four of the common bases of RNA as well as inosine and is found in a variety of positions on these compounds. In contrast, sugar methylation of RNA is found only at the 2' position of ribose although pentoses and hexoses methylated at other posi- tions are known to occur in compounds other than RNA (Bacon and Cheshire, 1971). The distribution of methylation between base and sugar varies depending on the RNA and the organism. In the ribosomal RNA of E. 221;, where the total methylation is about 0.7 mole %, some 17-23% of these methyl groups are present as sugar methylation (Hayashi et a1, 1966; Isaksson and Phillips, 1968). However, in yeast rRNA (Retel et a1, 1969) this figure rises to about 80% of the total methylation (1.1 mole %) while it reaches 90% in tissue cultured mammalian cells (Tamaoki and Lane, 1968; Vaughn et a1, 1967). The situation with transfer RNA is quite different since base methylation is always greater than sugar methylation. Whether in E, £211_(Hayashi et a1, 1966) where total tRNA methylation approaches 3% or in yeast or wheat germ tRNA (Gray and Lane, 1967) which are methylated to the ex- tent of 5-6%, the 2'-Qfmethylation amounts to only 15-25% of RNA methylation. Th confers both re mediate earlies stabili‘ Allen (1 amountir the conc room ten Q-methyl (1955) a Pancreas make it It was n alkali.S ation by dieStera NUCleosi groups nu differen tions we] NiCot‘ . % SOluble I Ye110w mc Historical The replacement of the 2'-hydroxy1 with a methoxy group confers on the RNA both alkali and ribonuclease stability since both reactions proceed through a cyclic phosphodiester inter- mediate involving the 2' and 5' positions of ribose. Thus, the earliest discoveries of 2'-Qfmethy1ation were related to alkaline stability. Smith and Allen (1953) and Crestfield, Smith and Allen (1955) reported an alkali-stable fraction in RNA from yeast, amounting to about 5 mole percent of total nucleotides. However, the conditions of alkaline hydrolysis, 1 M NaOH for 2h hours at room temperature, would not hydrolyze certain rather stable non- Qfmethylated linkages (Singh and Lane, 196hb). Potter and Dounce (1956) also reported an alkali-stable fraction in yeast, calf pancreas and rabbit liver RNA but the high values reported (5-58%) make it likely that DNA contamination contributed to these values. It was not until 1959 that Smith and Dunn demonstrated that alkali-stability could be most likely attributed to 2'-Qfmethy1- ation by hydrolyzing the alkali-stable fractions with snake venom diesterase and running borate chromatography on the products. Nucleosides with 2'-Qfmethyl groups do not have the cis-hydroxyl groups necessary to form a complex with borate and thus can be differentiated from normal nucleosides. Such alkali-stable frac- tions were found in RNA of wheat embryo and Beta vulgaris and Nicotiana glutiosa leaves, as well as rat liver microsomal and soluble RNA. No alkali-stable fractions were found in Turnip yellow mosaic virus or Aerobacter aerogenes RNA. In the years £011 and rat me tl tRN. be 1 altl am01 Chrc and Lam ato; et . 0f! 0f pho The following, 2'—Qfmethylcytidine was demonstrated in Anacystis nidulans RNA (Biswas and Myers, 1960), 2'-Qfmethy1cytidine and 2'-Qfmethylguanosine were recovered from yeast tRNA and rat liver RNA (Morisawa and Chargaff, 1963), and all four methylated nucleosides were found by Hall (1963a) in yeast tRNA. Thus, at this time 2'-Qfmethy1ated nucleotides could be taken to be an integral part of RNA from higher organisms although little quantitative data were available. 2'-0-Methyl Levels in Ribosomal and Transfer RNA Such data were soon forthcoming in the work of Ross Hall (1965b and 196M) and Singh and Lane (196ha and l96hb). The amount of 2'-97methylation present was measured by column chromatographic methods, both partition columns (Hall, 1963b) and DEAE-cellulose columns (Lane and Allen, 1961; Singh and Lane, l96ha) being used. These methods, as well as paper chrom- atography (Lane, 1965) and DEAE-Sephadex chromatography (Wagner et al, 1967; Diuer et a1, 1970) have been used by several groups of workers to determine the 2'-Qfmethy1 contents of rRNA and tRNA of a variety of species (Table I). A fact that should be noticed from this table is that the early work of Hall gives 2'-Qfmethyl values which are quite low compared to the results of later workers. In Hall's technique, which uses snake venom phosphodiesterase hydrolysis of RNA, any unhydrolyzed material would not be detected by the partition column chromatography. The RNA was not checked for complete hydrolysis and it is known lmlmlmlmlmlm . O O O Q C o o H H TABLE I 2'-O-METHYLATION LEVELS IN RIBOSOMAL AND TRANSFER RNA Mole % 2'-Q- RNA Methylation Reference .E- coli rRNA 0.1 Nichols and Lane, 1966a ‘E. coli rRNA 0.17 Hayashi et al, 1966 E. coli rRNA 0.12 Isaksson and Phillips, 1968 E. coli tRNA 0.22 Hall, 1961+ ‘E. coli tRNA 0.5 Nichols and Lane, 1966a E. coli tRNA 0.7 Hayashi et a1, 1966 Yeast (S. carlsbergensis) rRNA 1.1 Retel et a1, 1969 Chinese cabbage leaves rRNA 1.5 Dunn et a1, 1965 Wheat germ rRNA 1.7h Lane, 1965 Sheatfish liver rRNA 1.61 Diuer et al, 1970 Sheep liver rRNA 1.02 Hall, 196k Sheep heart total RNA 0.6M Hall, 196h L-cell rRNA 1.2 Lane and Tamaoki, 1969 Human liver rRNA 0.56 Hall, l96h HeLa cell rRNA 1.5 Vaughn et al, 1967 HeLa cell rRNA 1.h Wagner et a1, 1967 Novikoff hepatoma cell rRNA 1.5 Egawa et al, 1971 Yeast tRNA 0.62 Hall, 196k Yeast tRNA 0.8 Gray and Lane, 1967 Yeast tRNA 1.1 Morisawa and Chargaff, 1968 Wheat germ tRNA 1.50 Hudson et al, 1965 Sheep liver tRNA 0.58 Hall, l96h Calf liver tRNA 0.67 Hall, l96h Human liver tRNA 1.05 Hall, l96h Metastatic tumor liver 0.16 Hall, 196% (total RNA) Rhabdomyosarcoma 0.h1 Hall, l96h (total RNA) Mouse Ehrlick ascites 0.7h Hall, 196k (total RNA) Murphy-Sturm lymphosarcoma 1.01 Hall, 196h (total RNA) that linka the l conte C.l% seem: and 1 high« than VEI‘S inve cont weig all WEiE 1’RN. heel 13an nucl C111, that snake venom diesterase works only slowly on 2'-Qfmethy1 linkages (Gray and Lane, 1967). Therefore, this may explain the low values observed. The values in Table I clearly show that the 2'-Qfmethy1 content of RNA can vary quite widely over a range of about 0.1% to 1.7%. With the exception of Hall's early data, there seems to be a gap between the values for bacteria (E; ggli only) and higher organisms. In addition, it would seem that in higher organisms, ribosomal RNA is more highly 2'-Qfmethylated than the corresponding tRNA, while this relationship is re- versed in bacteria. Several of the rRNA Species listed in Table I have been investigated in greater detail. Table II shows the 2'-Qfmethy1 content of a number of rRNAs where the high and low molecular weight components have been separated. It can be seen that in all cases, with the exception of yeast rRNA, the low molecular weight rRNA is more highly methylated. Other RNA Species The work described thus far has been concerned with whole rRNA or tRNA. However, a large number of new RNA Species have been discovered recently. Some of these have had their 2'-Qf methyl content investigated. A small molecular weight RNA com- ponent of ribosomes, SS RNA, is known to contain no modified nucleotides from work giving the complete sequences of such mole- cules isolated from E. coli (Brownlee et al, 1967) and KB cells TABLE II 2'-Q:METHYLATION OF RIBOSOMAL RNA COMPONENTS Mole % rRNA 2'-97Methy1ation Reference ‘E. coli 258 0.15 Hayashi et al 163 0.20 1966 Yeast 268 1.2 Retel et al 173 1.0 1969 L-cell 283 1.1 Lane and Tamoaki 163 1.5 1969 HeLa cell 288 1.1-1.2 wagner et al 183 1.8 1967 HeLa cell 288 1.2 Vaughn et al 163 1.5 1967 Novikoff cell 288 1.29 Egawa et al 183 1.9M 1971 addit with later cular has a to de PUIse theSe into is pr Only tic s han b sugar methyl Cleotic (Forget and Weissman, 1967). Another ribosomal component is 78 RNA which is sometimes referred to as "288 associated RNA" (288 A RNA) and is found hydrogen bonded to the 28S rRNA of eucaryotic organisms. The molecule consists of some 150 nucleo- tides and in HeLa cells is methylated about one-third as much as 283 RNA or about 0.5% (Pene, Knight and Darnell, 1968). In addition, a number of nuclear and nucleolar RNA species are known, with S values ranging from 5 to 80. These will be dealt with later in sections pertaining to rRNA processing and small mole- cular weight nuclear RNA. Messenger RNA, both as cellular message and viral RNA, has also been examined. Moore (1966) used methyl-14C-methionine to demonstrate that no methyl label was incorporated into E, 22;; pulse labeled RNA or into the RNA of the viruses Th and R17 when these infected E, 221; cells. Also, poliovirus infection of HEp2 cells did not result in the incorporation of methyl label into virus RNA (Grado et al, 1968). Therefore, if 2'-Qfmethy1ation is present in messenger molecules in these organisms, it is in only extremely small quantities. However, mRNA from an eucaryo- tic Species has yet to be investigated. Distribution of 2'—0-Methy1ation In addition to looking at total 2'-Qfmethy1 content, there have been a number of reports which detail the distribution of the sugar methylation. Because of the alkali stability of the 2'59- methyl linkage, alkaline hydrolysis gives rise to as many dinu- cleotides as there are 2'-Qfmethyl groups present. Trinucleotides are p} Singh dinuci the m: found Conta trinu “0 tr YEast with alsO are produced if there are two adjacent 2'-Qfmethy1 linkages. Singh and Lane (196hb) first demonstrated that all 16 possible dinucleotide sequences were present in wheat germ rRNA with the most common being AmpGp (11%) and AmpAp (9%). They also found that about 10% of the total alkali-stable material was in the form of trinucleotides which is approximately three times more than would be expected on a random basis. Seven such trinucleotides were found including Ummep 1p, which incorporates a pseudouridine residue (Lane, 1965). Wheat germ tRNA in contrast contains only 15 of the normal dinucleotides in addition to two which havelhn as their methylated half. Here, the most common dinucleotides were CmpUp (20%) and GmpGp (18%). No trinucleotides were found. A similar situation is found in yeast, where the rRNA contains all 16 2'-Qfmethyl containing dinucleotides and three trinucleotide sequences while the tRNA possesses only 11 with no trinucleotides (Gray and Lane, 1967). Another study of yeast tRNA (Morisawa and Chargaff, 1968) found 12 dinucleotides with GmpGp predominating (55.5%). All 16 dinucleotides have also been found in liver rRNA of the Sheatfish (Silurus glanis), a relative of the catfish (Diuer et a1, 1970). In the cases of L-cell rRNA (Tamaoli and Lane, 1968), HeLa cell rRNA (wagner et a1, 1967), Novikoff hepatoma cell rRNA (Egawa et a1, 1971) and E, ggll_rRNA (Nichols and Lane, 1966b), the dinucleotide sequences have been elucidated separ- ately for the high and low molecular weight rRNAs. The three studies of the rRNA of the eucaryotic cells have shown that the are Sing dinu the! founl time: such inc01 Conta trinu n0 tr Yeast with also- a reh HeLa < rRNA 1 1966b) ately Studie are produced if there are two adjacent 2'-Qfmethy1 linkages. Singh and Lane (196kb) first demonstrated that all 16 possible dinucleotide sequences were present in wheat germ rRNA with the most common being AmpGp (11%) and AmpAp (9%). They also found that about 10% of the total alkali-stable material was in the form of trinucleotides which is approximately three times more than would be expected on a random basis. Seven such trinucleotides were found including Ummep hp, which incorporates a pseudouridine residue (Lane, 1965). Wheat germ tRNA in contrast contains only 15 of the normal dinucleotides in addition to two which have hm as their methylated half. Here, the most common dinucleotides were CmpUp (20%) and GmpGp (18%). No trinucleotides were found. A similar situation is found in yeast, where the rRNA contains all 16 2'—Qfmethyl containing dinucleotides and three trinucleotide sequences while the tRNA possesses only 11 with no trinucleotides (Gray and Lane, 1967). Another study of yeast tRNA (Morisawa and Chargaff, 1968) found 12 dinucleotides with GmpGp predominating (55.5%). All 16 dinucleotides have also been found in liver rRNA of the Sheatfish (Silurus glanis), a relative of the catfish (Diuer et a1, 1970). In the cases of L-cell rRNA (Tamaoli and Lane, 1968), HeLa cell rRNA (wagner et a1, 1967), Novikoff hepatoma cell rRNA (Egawa et a1, 1971) and E, 221; rRNA (Nichols and Lane, 1966b), the dinucleotide sequences have been elucidated separ- ately for the high and low molecular weight rRNAs. The three Studies of the rRNA of the eucaryotic cells have shown that the dinucl weight to cor rRNA < cleot: (Wagn: the m the w three and U (N4Me also the u dist] Wide inve: the~ is k Work net noti tide Peti dinucleotide patterns differ between the high and low molecular weight components. The L-cell and Novikoff cell Species seem to contain all the possible dinucleotides while HeLa cell 28S rRNA contains only 11 sequences and the 18S rRNA has 15 dinu- cleotides. TWO trinucleotides are also found in the 28S rRNA (Wagner et a1, 1967). In the case of the rRNA of E, 221}, where the methylation level is far below that in mammalian cells, the work of Nichols and Lane (1966a and b, 1967) has demonstrated three principal dinucleotides in the 25S rRNA (GmpGp, CmpCp and UmpGp) and only a single major sequence in the 168 rRNA (N4MeCmpCp). The same workers (Nichols and Lane, 1967) have also discovered five dinucleotide sequences in E, 321; tRNA, the most common being GmpGp (58%), CmpUp (50%) and umpUp (28%). It is difficult to come to any conclusions regarding the distribution of dinucleotide sequences except that there is a wide variety of patterns in the RNA molecules which have been investigated. In addition, rRNA is more likely to contain all the possible dinucleotide sequences than is tRNA and only rRNA is known to contain trinucleotides. It is also now known from work elucidating the total sequences of several tRNA species that not all tRNA molecules contain a 2'-Qfmethyl linkage (Madison, 1969). There is one peculiarity of distribution which deserves notice: in all the RNA Species thus far studied, the dinucleo- tide GmpGp has been either the major sequence or a close com- petitor. This is true for rRNA and tRNA and for E, ggll_as well as the eucaryotic organisms. An investigation of the bases associated with the methylated dinucleotide patterns differ between the high and low molecular weight components. The L-cell and Novikoff cell Species seem to contain all the possible dinucleotides while HeLa cell 28S rRNA contains only 11 sequences and the 188 rRNA has 15 dinu- cleotides. TWO trinucleotides are also found in the 28S rRNA (Wagner et a1, 1967). In the case of the rRNA of E, 2211, where the methylation level is far below that in mammalian cells, the work of Nichols and Lane (1966a and b, 1967) has demonstrated three principal dinucleotides in the 25S rRNA (GmpGp, CmpCp and UmpGp) and only a single major sequence in the 163 rRNA (N4MeCmpCp). The same workers (Nichols and Lane, 1967) have also discovered five dinucleotide sequences in E, 221; tRNA, the most common being GmpGp (58%), CmpUp (50%) and DmpUp (28%). It is difficult to come to any conclusions regarding the distribution of dinucleotide sequences except that there is a wide variety of patterns in the RNA molecules which have been investigated. In addition, rRNA is more likely to contain all the possible dinucleotide sequences than is tRNA and only rRNA is known to contain trinucleotides. It is also now known from work elucidating the total sequences of several tRNA Species that not all tRNA molecules contain a 2'-Qfmethyl linkage (Madison, 1969). There is one peculiarity of distribution which deserves notice: in all the RNA Species thus far studied, the dinucleo- tide GmpGp has been either the major sequence or a close com— petitor. This is true for rRNA and tRNA and for E, 221; as well as the eucaryotic organisms. An investigation of the bases associated with the methylated 10 sugars has been made in many cases and Table III summarizes some data on the distribution of 2'-Qfmethyl groups among the various nucleosides. In general, Gm and Cm seem to be found in excess in tRNA while Am is quite rare. In rRNA from higher or- ganisms there seems to be a more even distribution among the four nucleosides. This is further borne out in studies with rRNA where all the dinucleotides were not resolved so that only estimates of the total distribution can be made. This is the case with L-cell, Novikoff cell and Sheatfish liver rRNA which all show a fairly even distribution. Problematical Cases Before leaving the area of general 2'—Qfmethyl content, two organisms must be dealt with where sharply conflicting reports have been published. Saprospira grandis WH, a flexi- bacterium, contains cylindrical bodies called rhapidosomes. Correll and Lewin (l96h) first reported that the RNA from these rhapidosomes was at least 85% 2'-Qfmethylated. In a later paper, Correll (1968), using alkaline hydrolysis and DEAE- cellulose chromatography, examined seven strains of flexibac- teria for sugar methylation of their rhapidosomal RNA. He reported levels of h0-95% for 2'-Qfmethy1ation in these strains. The flexibacterial rRNA and tRNA had methylation values below 5%. However, two separate laboratories have Since published papers on SaprOSpira rhapidosomes which do not support these results (Delk and Dekker, 1969; Price and Rottman, 1970a). HHH NQQAWH. 11 a fiw wafiwsfiocH .80 wow E< awesome vopw>wp mfiaosuo on OS Afidv uoam aH0moucs onu mafianmm< Somfi .Hamm Pmmfi .ocmq was home wwma «mmmwuwzo paw m3wmwuoz meH «coma can mono Somfi .aamm ewma .msmq was maoeoaz zoma .aamm swma .Samm Emma .Hm um uoawmz wwma .ocma paw zmuu eoma .mcmo Sam mfioeosz mocoummmm mm :m ma P OH mm pm mm ma mm PH ED Hm mm mm mm m: *mm :4 mm an ma :0 80 mm an m: 6n oe an pm pm mm om ma BU ma 4.0 m J m *m m mm mm on E< coHumfixsuSZLmn.m HMDOH & «ZMu uo>w~ amonm wa moonm <2m~ HHoo mgom EHMZLW:.N ho ZOHHDmHMHmHQ HHH mqm<fi Neit acco: methyl fOUnd in the and Sp. 1969) . 8° that 12 Neither RNA purified from rhapidosomes nor RNA prepared according to Correll (1968) showed more than 5% alkali-stable material. Another class of organisms from which variable results have been obtained is Mycoplasma, organisms smaller than bac- teria and free living. Hall et a1 (1967) first reported that in total RNA of Mycoplasma strain 880, they could not find any modified nucleosides using techniques which should have been able to detect sugar methylation at the 0.1% level. Mere recently, Hayashi et a1 (1969) investigated the tRNA of Myco- plasma laidlawii B and the Kid strain of MycoPIasma and were able to Show the presence of 2'-Qfmethylnucleosides as well as pseudouridine, ribothymidine, h-thiouridine and several base methylated species. No quantitative data were given but the amounts of minor nucleotides were said to be less than in E, coli. These data became available in 1971 when Feldman and Falter used 32? labeling and thin layer chromatography to study minor nucleotides in the tRNA of Myc0plasma laidlawii A. They determined a value of about 1% for 2'-Qfmethylation while base methylation amounted to about 5.5%. Pseudouridine was also found to the extent of 5%. Thus, there are large discrepancies in the amounts of minor nucleotides found in various strains and Species of Mycoplasma. However, a recent review (Hayflick, 1969) stresses the heterogeneity of the group Myc0plasmatales so that the organisms studied may not be comparable. 15 Low Molecular weight Nuclear RNA In addition to the cytoplasmic RNA Species thus far considered, there is a large group of both high and low mole- cular weight types of RNA which are found in the nucleus of eucaryotic cells and which represent about 2% of the total cellular RNA. The nucleoplasm contains a class of very high molecular weight heterogeneous RNA (McCarthy et a1, 1970) as well as the 28S and 188 precursors to cytoplasmic rRNA. In addition, there exists a group of low molecular weight RNAs which sediment at h-8S (Dingman and Peacock, 1968). Included in this group are the nuclear predecessors to 58 rRNA and 288 A RNA. The nucleolus contains the high molecular weight precursors to ribosomal RNA (#58, #18, 528, 288, and 208) which are involved in RNA processing. Also present are the h-88 Species, again including 58 rRNA and 288 A RNA, which are synthesized in the nucleolus. Although both the nucleus and nucleolus contain the low molecular weight species, their complements are not the same, since there exist Species which are peculiar to the nucleolus as well as species common to both organelles (wein- berg and Penman, 1968). Two of these small RNA Species appear to be hydrogen bonded to nucleolar 28S RNA though neither are found associated with cytoplasmic 28S rRNA (Prestayko et al, 1970). The nuclear heterogeneous RNA has not been well char- acterized although some recent evidence points to its being a precursor to mRNA (Darnell et al, 1971). The status of methyl- ation in these molecules is uncertain. The rRNA precursor mole: be t of t et a when rapi ber 197:5 even 1969 by ti 19C: 1h molecules and the role of methylation in rRNA processing will be the subject of the next section. The low molecular weight species, which represent 15-20% of the total nuclear RNA (weinberg and Penman, 1968; Moriyama et al, 1969), are unusual in being metabolically quite stable wheras the remainder of the nuclear RNA turns over with great rapidity (Weinberg and Penman, 1968 and 1969). The exact num- ber of Species is unknown but is at least ten (Ro-Choi et a1, 1970), and these give similar gel electrophoretic patterns even when isolated from different Species (Rein and Penman, 1969). These molecules have been shown to be highly methylated by their uptake of methyl-14C-methionine (weinberg and Penman, 1968; Moriyama et al, 1969; Zapisek et al, 1969). Weinberg and Penman (1968) have estimated the total methyl content of these Species at about 2-h% and alkaline hydrolysis followed by separation on a DEAE-cellulose column has identified much of the methyl label as 2'-Qfmethy1ation (Zapisek et al, 1969). These latter workers estimate the total methylation at 50-60% of the total methylation of #8 RNA (which is about h-6%) but give no quantitation as far as 2'-Qfmethylation is concerned. One of the small nuclear RNAs from Novikoff hepatoma cells (h.58 RNA III) has been isolated and nucleotide analysis has shown the presence of four dinucleotides (AmpAp, GmpAp and two GmpGp) as well as a 2'-Qfmethyluridine 5'-terminus. These five sugar methylated compounds among 85 nucleotides amount to nearly 6%. ye me ce 00 C3. the car pre is tir met pri 0f val the; intc to p rate 15 Ribosomal RNngrocessing (Eucaryotic Organisms) The subject of rRNA processing in the nucleolus and nucleus has been one which has received a great deal of study in recent years. One point of Special interest has been the role of methylation in the processing. Labeling of HeLa cells with methyl labeled 14C-methionine showed that the h58 molecule was first methylated and that this label can be chased into 528, 288 and 188 rRNA (Greenberg and Penman, 1966; Zimmerman and Holler, 1967). A scheme which has been proposed for the pro- cessing of h58 RNA in HeLa cells is Shown in Figure 1. It is obvious that much of the h58 RNA molecule (about 50% in this case) is lost in the processing. In other organisms there are differences in the sizes of the molecules involved, but the general pattern seems to be the same. This normal scheme can be interrupted by depriving the cell of methionine, the precursor for both the base and sugar methyl groups. If this is done (Vaughn et al, 1967), #58 and 52S RNA synthesis con- tinues but there is no production of 288 or 188 RNA. In such methionine deprived cells the 2'-Qfmethy1 content (which com- prises Some 90% of the total methylation in the #58 and 52S RNA) of the nucleolar RNA drops to about 1/5 to 1/5 of the control value. Further, if methionine is added to deprived cells, there is an immediate incorporation of labeled methyl groups into the undermethylated RNA and processing of the RNA returns to normal. Only normal processing is seen, although at a lower rate, if the cells are starved for an amino acid other than 16 TABLE IV 2'-QfMETHYLATION AND rRNA PROCESSING IN NOVIKOFF HEPATOMA CELLS Total % Total RNA Number of Alkali-stable 2'-97Methyl- Nucleotides Dinucleotides ation Nucleolar Ass 12800 115 0.89 Nucleolar 558 7800 82 1.10 Nucleolar 288 5700 66 1.16 Ribosomal 283 A500 56 1.29 Nucleolar 258 5200 57 1.17 Ribosomal 183 1900 59 1.9M meth buta mole grou in t the ment rRNA cons Cleo RNA 188 meth RNA Will org; larg Port Con: 8r01 the and 17 methionine, so this would seem to be an effect directly attri- butable to undermethylation. All methylation seems to occur at the level of the #58 molecule with a single exception in HeLa cells. The methyl groups on the base of Ne-dimethyladenosine which is found only in the 18S RNA appear to be added at the time of cleavage of the #58 molecule (Zimmerman, 1968). In HeLa cells, measure- ments of 2'jQ-methylation in the nucleolar precursors and the rRNA Show that all the methyl groups of the #58 molecule are conserved (Vaughn et al, 1967), as do experiments where dinu- cleotide patterns of methyl-3Hdmethionine labeled nucleolar RNA are compared with methyl-14C-methionine labeled 288 plus 188 RNA (Wagner et a1, 1967). Work also using methyl-14C- methionine label which compares T1 digest fingerprints of #58 RNA versus 288 plus 188 RNA again tells the same story (Salim, Williamson and Maden, 1971). This is not the case for other organisms, however. In yeast (Retel et al, 1969) there is a large amount of secondary base methylation and the degraded portions of the #58 molecule are as highly methylated as the conserved parts. Another example of non-conservation of methyl groups occurs in the Novikoff hepatoma cell. Table IV shows the results of work by Choi and Busch (1970) and Egawa, Choi and Busch (1971). In the scheme proposed, #58 RNA gives rise to 558 RNA and 25S RNA, these two being respectively the precursors of 28S and 188 RNA. No methyl groups appear to be lost in the f1) 18 FIGURE 1 PROCESSING OF #58 NUCLEOLAR RNA (Weinberg and Penman, 1970) A53 (h.1 x 106 MW) #1S (5.1 x 106) (1.0 x 106) (degraded) 528 (2.1 x 106) ‘r”’/#L\\\\ii 208 (0.95 x 106) C— 283 (1.65 x 106) (0.h5 x 106) (0.5 x 106) 183 (0.65 x 106) (degraded) (degraded) 78 (0.0# x 105) 19 first cleavage of #58 RNA but in the processing of 558 RNA to 28S rRNA, there is a loss of some 26 2'-Qfmethyl groups. Thus, two of the Steps involve loss of unmethylated pieces of RNA while one step results in the loss of a piece which is nearly as highly methylated as the rest of the molecule. An interesting observation with regard to processing in Novikoff cells is that the #58, 558 and 28S molecules all have 2'-Qfmethylcytidine as the 5'-terminus. This means that the 55S and 288 molecules are most likely derived from the 5'-end of the #58 RNA (Choi and Busch, 1970). Ribosomal RNA Processing (Procaryotic Organisms) Although the phenomenon of rRNA processing in bacteria is less complex than in the eucaryotic organisms, there is a simi- larity in the existence of precursor molecules. Both kinetic data and high resolution gel electr0phoresis point to the pre- sence of precursors for the 25S and 168 rRNA of both E. ggll_ (Adesnik and Levinthal, 1969; Pace, Peterson and Pace, 1970) and E. subtilis (Hecht and Woese, 1968). Maturation of these precursor molecules can take place even in the presence of Actin- omycin D, which prevents further RNA synthesis. A low molecular weight precursor to the 58 rRNA of E, 22;; also exists (Adesnik and Levinthal, 1969), but in E, subtilis the precursor seems to be of high molecular weight (Hecht, Bleyman and Woese, 1968) and 58 rRNA in this case may represent a piece cleaved off the 25S RNA precursor since the 258 and 58 genes are linked (Colli, Smith and Oishi, 1971). Whatever may be the ancestry of 58 RNA 20 there is no evidence for a concerted synthesis of 25S and 16S rRNA by means of a single precursor such as is found in higher organisms, although all three rRNA genes are linked (Colli et al, 1971). Treatment with Chloramphenicol (CAM) or starvation for an essential amino acid in a relaxed strain results in the formation of immature particles which contain precursor rRNA. The proteins associated with the rRNA in these particles is non-ribosomal protein which existed in the cell at the time of protein synthesis blockage so that the particles do not represent immature ribosomes (Yoshida and Osawa, 1968; Schleif, 1968). The precursor rRNA in CAM.particles is undermethylated to the extent of 50-60% in the 25S RNA and 80-90% in the 168 RNA (Hayashi et al, 1966; Dubin and Gunalp, 1967). Specif- ically, the 2'-Qfmethy1 content decreases from 0.15% to 0.10% in the 25S RNA and from 0.20% to 0.015% in the 16S RNA. Thus, the 2'-Qfmethyl fraction of the total methylation is higher in CAM 25S RNA and lower in CAM 168 RNA (Hayashi et a1, 1966). In the case of bacteria, any inhibition of protein synthesis will result in the formation of methyl-poor rRNA precursor so that the situation is not similar to that in higher organisms. It is likely that maturation of bacterial rRNA involves methyl- ation of the molecule over a period of time instead of all at once in a precursor molecule. Therefore, any agent which pre- vents maturation will produce undermethylated RNA. In any case, methylation plays a role in bacterial rRNA maturation as it does in the processing of eucaryotic rRNA. 21 The Effect of 2'—0-Methylation on Enzymes of RNA Metabolism Aside from the inhibitory effect on pancreatic ribonuclease mentioned earlier, only a few reports are available regarding the effects of sugar methylation on enzyme action. It is known that 2'-Qfmethyl nucleotides are not dephOSphorylated by 5'-nucle- otidase from either bull semen or snake venom (Honjo et al, l96#) and that dinucleotides with 2'-Qfmethy1 linkages prove to be refractory to hydrolysis by purified Russell's viper (Vipera russelli) venom ph03phodiesterase (Gray and Lane, 1967). In contrast, acetylation of the 2'-hydroxy1 position does not affect Central Asian viper (Vipera lebetina) venom phosphodiesterase while completely blocking the action of spleen diesterase (Knorre et al, 1966), an enzyme not affected by the presence of a 2'-Qfmethyl group. In the case of another enzyme involved in RNA metabolism, it has recently been shown (Gerard, Rottman and Boezi, 1971) that 2'-Qfmethyladenosine 5'-triphosphate can act as a substrate for bacterial DNA-dependent RNA polymerase although further addition to the RNA chain is greatly inhibited by this incorporation. Synthetic 2'-O-Methy1 Compounds Although Szer and Shugar (1961) reported the synthesis of N3,2'-Qfdimethyluridine in a 10% yield using 2',5'-insopropy1- idene uridine and excess diazomethane, the first direct prepara- tion of a 2'-Qfmethylnucleoside was performed by Broom and Robins (l96#). They reacted adenosine with an aqueous solution of diazome Under 1 prefere produce methyll 2'-Q-m« Recent' silver 22 diazomethane and obtained a #1% yield of 2'-Qfmethyladenosine. Under the conditions used, the 2'-hydroxy1 position reacts preferentially although some 5'-Qfmethylated compounds are also produced. The same conditions can be used to prepare 2'-Qf methylcytidine and deamination of this compound will yield 2'—Qfmethyluridine (Martin, Reese, and Stephenson, 1968). Recently a modified method using diazomethane and stannous Silver chloride dihydrate has been used to prepare the 2'- and 5'-Qfmethyl derivatives of adenosine and cytidine (Robins and Naik, 1971). It has not proved possible to synthesize 2'-Qf methylguanosine in the simple manner of the nucleosides. In- stead a several step procedure is necessary with diazomethane again being the methylating agent (Khwaja and Robins, 1966). Rottman and Heinlein (1968) first Showed the incorporation of a 2'-Qfmethylated compound into a polymer by using poly- nucleotide phosphorylase and AmDP to make Poly (Am) under conditions of high enzyme concentration. These conditions also suffice for the synthesis of Poly (Um) but Poly (Cm) synthesis requires the substitution of manganese for magnesium (Janion, Zmudzka and Shugar, 1970). Heteropolymers containing a methylated and unmethylated component have also been prepared (Rottman and Johnson, 1969). In these polymers, the 2'-Qfmethyl- nucleotides occurred in pairs in much greater than random fre- quency, but a high degree of randomness could be achieved in Poly (AmC) but not in Poly (CmU) by use of dimethylsulfoxide (Rottman and Johnson, 1969). 25 Speculations on Function Although no complete explanation can be given of the function of 2'-Qfmethylnucleotides in RNA, some possibilities can be examined. It is obvious from the data given above that 2'-Qfmethylation is extremely wideSpread, being found in all manner of organisms and in nearly all classes of RNA. However, it is not ubiquitous, 58 rRNA and several Species of tRNA contain no 2'jgdmethyl linkages and it is probable that mRNA and viral RNAS are likewise devoid of sugar methylation. In addition, studies with methyl-deficient tRNA obtained from methionine starved relaxed auxotrophs demonstrate that methyl- ation is not a sine qua non for tRNA function in amino acid incorporation (see references in Starr and Sells, 1969). As for positive statements, there does appear to be a strong link between methylation and rRNA processing as is evi- denced by the results of methionine Starvation in HeLa cells. There is the possibility that the methylation pattern may act as a recognition signal for the nuclease which cleaves the #58 RNA molecule. In addition, there seems to be a corres— pondance between the extent of 2'-Qfmethylation and stability of RNA. The extreme stability of such highly methylated molecules as the low molecular weight nuclear RNAs can be con- trasted with the extreme lability of the rest of the nuclear RNA and of messenger RNA. In rRNA processing as well, it is usually the unmethylated or undermethylated segments of the #58 RNA which are degraded. Experiments with synthetic 21- P01 the hon Fri ina in ril the 112 fat as of dit met 2'1 Stag meth boli acCo: for 0 tests 2h 2'-Qfmethylated heteropolymers also give similar results. Poly (AmC) and Poly (CmU) were found to be degraded more slowly than their unmethylated counterparts when incubated in a cell homogenate supernatant containing a variety of nucleases (Dunlap, Friderici and Rottman, 1971). On the other hand, such molecules are not biologically inactive; both Poly (AmC) and Poly (CmU) can act as templates in a cell-free amino acid incorporating system (Dunlap et al, 1971). In addition, 2'-Qfmethylated oligonucleotides have been shown to be competent as templates for the binding of tRNA to ribosomes (Price and Rottman, 1970b). Thus, it is possible that sugar methylation may be a cellular mechanism for stabi- lizing certain sequences of RNA. However, this all remains as conjecture with only scattered facts for support. In the study of a modification as widespread as 2'-Qfmethy1ation, it is of importance to determine the extent of methylation in different organisms and under various con- ditions. A knowledge of the natural distribution of sugar methylation is a necessary condition to understanding its bio- logical function. Therefore, in this thesis, an investigation is made of 2'-Qfmethylation levels in organisms at several evolutionary stages. A study is also made of possible changes in 2'79- methylnucleotide content in the RNA in periods of intense meta- bolic activity or gross physiological change. In order to accomplish these goals, a more sensitive and accurate technique for determining 2'-Qfmethy1ation is developed and thoroughly tested. 25 METHODS Determination of 2'-0-Methylation by Gas Chromatography As has been mentioned, column chromatography has been the main method used for the determination of 2'jQ-methylation. If a radio- active label can be introduced, paper chromatography can be pro- fitably utilized. Both these techniques are unsuitable for the assay of a large number of samples, the former being laborious and requiring a large sample and the latter being useful only when a radioactive label can be incorporated into the RNA. In many cases of interest, only a small amount of RNA is available and radioactive labeling is not feasible. Also, in bacteria the low amount of sugar methylation makes very large amounts of material necessary. Thus, there was an obvious need for a technique which would be specific for sugar methylation and would require only small amounts of RNA for analysis. Treatment of methylated pentoses with strong acid gives rise to furfural and methanol (Bott and Hirst, 1952). Baskin and Dekker (1967) first used this fact to quantitate sugar methylation by micro- distillation of perchloric acid treated labeled RNA. We have adapted this technique for use with small amounts of non-labeled RNA. The method and the controls used are described in the following section in the format of a paper which has been accepted for publication in Analytical Biochemistry but is still in press. A Gas Chromatographic Method for the Determination of 2'-O-Methy1ation in RNA1 Joseph Abbate and Fritz Rottman Department of Biochemistry Michigan State University East Lansing, Michigan #8825 Running Title: Determination of 2'-0-Methylation This Scie med: Agr: uni tes pea FOOTNOTES This work was supported by Grant GB-2076# from the National Science Foundation and by a Michigan State University Bio- medical Sciences Support Grant 5-805-FR070#9-0#. Michigan Agricultural Experiment Station Journal No. 5507. we found that different batches of Porapak 0 did not behave uniformly in these studies. Various lots of Porapak Q were tested until adequate separation of the methanol and furfural peaks was obtained. known Methyl while posit: direC‘ which lated methyi in RN; therm. radio. tativ. 2'-me‘ Perchf diSti of th be us. analy, emploj by th P0331 methy INTRODUCTION Ribonucleic acid (RNA) isolated from various organisms is known to be methylated in both the base and sugar moieties (1). Methylation of the bases may occur at any of several positions while sugar methylation is known to occur only at the 2'-hydroxyl position. In certain studies (2), we have found it necessary to directly determine the amount of sugar methylation by a method which is both sensitive and unaffected by the presence of methy- lated bases. While one can use the incorporation of radioactive methyl-labeled methionine to determine the amount of methylation in RNA, this method is not Specific for sugar methylation. Fur- thermore, it is not always possible or convenient to introduce radioactive methyl precursors. Baskin and Dekker (5) have described a method for the quanti- tative determination of sugar methylation in RNA in which the 2'-methoxy group is released as methanol by digestion with 70% perchloric acid. The methanol is generally determined by micro- distillation (5,#). If microdistillation is used for quantitation of the released methanol, only radioactively labeled material can be used. we have adapted this perchloric acid technique for the analysis of semi-micro amounts of non-radioactive RNA samples by employing gas chromatography to quantitate the methanol produced by the hydrolysis. we have also eXplored the applications and possible limitations of this technique in determining the 2'-0- methyl content of RNA samples. unmc B101 fro: salt DL-C pur: t01 gert (Bu< Shee 0f . Scr Bio 8 am RNA 3mm MATERIALS AND METHODS The materials used were obtained from the following sources: unmodified nucleosides and nucleoside monophosphates from Schwartz Bioresearch; nucleoside diphOSphateS and synthetic homopolymers from P-L Biochemicals; ribose, 5-0-methy1-D-glucopyranose and salmon Sperm DNA from Calbiochem; adenine, thymine, deoxy-AMP, DL-O-methylserine, O-methyl-L-tyrosine and the base methylated purine and pyrimidine derivatives from Sigma Chemical Co. Wheat germ ribosomal and transfer RNA were prepared according to the procedure of Singh and Lane (5) using non-heat-treated wheat germ. Yeast tRNA was prepared from fresh cakes of Baker's yeast (Budweiser) according to the procedure of Holley 35 El: (6). The sheep liver RNA was purified as described by Hall (7) using livers of freshly slaughtered animals. E, ggli_rRNA was prepared as de- scribed by Asano (8); E, 2211 tRNA was obtained from Schwartz Bioresearch. In all preparations of RNA, redistilled phenol was used and samples were extracted twice with phenol. As a final step, the RNA samples were subjected to the 2-methoxyethanol-cety1trimethy1- ammonium bromide treatment described by Bellamy and Ralph (9). This procedure removes acid and neutral polysaccharides as well as residual protein (10). Samples were analyzed for DNA contamination by the diphenylamine method (11). The amount of RNA present in the samples used for gas chromatography was determined by inorganic phosphate analysis of ashed samples (12). an 0V were While of ap tubes again (PH 3 the g KOH t are r anol Seale' at 4?, Sent metha Perch Preparation of Samples for Gas Chromatography Samples of nucleic acids or nucleic acid components (50 to 1000 pg in 5 to 250 pl) were placed in 0.5 x 9 cm pyrex glass tubes which were sealed off at one end. These tubes had been previously washed according to the following regimen: 12-15 hours in aqua regia, 15 minutes in boiling detergent, 15 minutes in boiling water, fol- lowed by several washes in distilled water. The nucleic acid samples were frozen and lyophilized in the tubes after which 50 ul of 70% H0104 (J. T. Baker Chemical Co.) was added and the tubes centrifuged in a clinical centrifuge. The tubes were then sealed and placed in an oven for 90 minutes at 110 C. After this hydrolysis, the tubes were cooled to room temperature and then placed in powdered dry ice. While the contents remained frozen, the tubes were opened and #0 ul of approximately 10 N KOH was added to the upper portion of the tubes. The quantity of KOH was previously determined by titrating against 70% H0104 to give a final solution which is Slightly basic (pH approx. 8-10) since acid rapidly destroys the Porapak used in the gas chromatography. Care must be taken in the addition of the KOH that mixing with the HClO4 does not take place before the tubes are resealed since the neutralization reaction is violent and meth- anol can be lost. After addition of KOH, the tubes were once again sealed, mixed on a Vortex mixer, and then centrifuged at 20,000 x g at H3O for 15 minutes. After centrifugation, two layers were pre- sent in the hydrolysis tubes; an upper liquid layer containing the methanol and a precipitate consisting of the tar produced by the perchloric acid digestion and the KC104 produced by the neutrali- 1 12.1! 1 zation. With the tubes placed in ice, a measured volume of the upper liquid layer was removed with a Hamilton syringe. Usually about 55 ul was obtained free of precipitate. These samples were transferred to another set of glass tubes which were placed in dry ice. To these samples, 5 ul of a 0.# ug/ul solution of n-propanol was added with a calibrated Lang-Levy micropipette. The n-propanol acts as an internal standard in the gas chromatography. At this point, the samples were usually analyzed on the gas chromatograph. However, they can be sealed and stored at -20 C for later chromato- graphy. Gas Chromatography Gas chromatography was carried out on a Hewlett-Packard F and M #02 gas chromatograph with a flame ionization detector. A six- foot glass column was packed with Porapak Q 100-120 mesh (Waters Assoc.).2 The column was run at 150 C and the Size of the sample injected was 5 ul. High purity nitrogen was used as the carrier gas at a flow rate of 55 cc/min. A typical separation is shown in Fig. 1. A methanol/n-propanol ratio was obtained by cutting out the area under each peak and weighing the chart paper on an analytical balance. Two separate methods were used to calculate the mole-percent of 2'-0-methyl in the samples. In the first method, a known amount of synthetic 2'-0-methyladenosine (15) was used as a standard and was carried through the entire procedure described above. The amount of methanol produced by this known standard was set as 100% 1:! \x r u I . 311.. I .I 1' 3 ' 7 ii I .4: .t ' ,. u: ll! lei ‘ methylation and the values for the other samples were calculated accordingly. The second method did not necessitate the use of a 2'-0-methy1- adenosine standard. However, two correcting factors had to be obtained, both of which were eliminated by the use of the 2'-0- methyladenosine Standard. First, since the methanol/n-propanol detector response ratio varied with changes in the intensity of the detector flame, it was necessary to directly inject a standard methanol-n-propanol solution and obtain a response ratio for each series of samples. Second, it was found that a fraction of the methanol was lost in the precipitate of tar and K0104 products in the hydrolysis procedure. This loss remained constant as long as the conditions of the procedure were reproduced. In order to cor- rect for this loss, a standard methanol sample was carried through the entire procedure to determine a recovery factor (F). Uhder the conditions described above, the recovery of standard methanol amounted to about 60%. This factor is dependent on the ratio of the liquid layer to the precipitate layer, which, in this instance, has been kept small so as to give high sensitivity with a small sample volume. Higher recovery values can be obtained by increasing the ratio of liquid layer to precipitate. Upon determination of the rSSponse ratio and the recovery factor, the calculation of percent methylation was made by the equation: Elx R x F P = mole % methylation us RNAZ MW where propa previ stand of tl base of me anal) and 1 compc fEre RNA 1 can 1 HClO, anal) Sides hOmOp PhOry were ribOS where M and P are the areas in arbitrary units of the methanol and propanol peaks, respectively, of the experimental sample; R is the previously determined response ratio of propanol to standard methanol; F is the recovery factor; i.e., the reciprocal of the fraction of standard methanol recovered; and MW is the average molecular weight of the nucleotide residues in the RNA sample examined. RESULTS Non-2'-0-Methylated Compounds As can be seen from Table l, determinations carried out on base methylated purine and pyrimidine compounds showed no release of methanol. Therefore, base methylation does not interfere with analysis of sugar methylation, confirming the results of Baskin and Dekker (5) and Retel E£.El° (#). Table 2 shows that deoxyribose compounds also did not produce methanol. Thus, DNA does not inter- fere with the assay except in the quantitation of the amount of RNA present. Nevertheless, the presence of DNA in large amounts can result in small supernatant fractions following hydrolysis with H0104 and make it difficult to obtain the necessary volume for analysis. The group of ribose compounds tested (Table 2) included nucleo- sides, nucleoside monophOSphates, nucleoside diphosphates, and both homopolymers and heteropolymers synthesized by polynucleotide phos- phorylase. Rather unexpectedly, in all cases, the values recorded were above the lower limits given by the free bases and the deoxy- ribose compounds. In order to confirm that these values do, indeed, represent detection of 2'-0-methyl groups in the samples tested, the following experiment was carried out with CDP, which was chosen because of the high values obtained for 2'-0-methy1 content (Table 2). The CDP was dephosphorylated with E, ggll_alkaline phosPhatase to give cytidine which was then placed on a Dowex l-X# column in the borate form to separate 2'-0-methylcytidine from cytidine (l#). Since 2'-0-methylnuc1eosides do not form a borate complex, they are eluted first with 0.5 M ammonium borate while the normal nucleosides are eluted with 0.7 M boric acid. Upon gas chromatography, the boric acid fraction was found to contain 0.1% 2'-O-methylnucleosides while the original CDP was methylated to the extent of 1.2%. The ammonium borate fraction was examined by paper chromatography and the isolated nucleoside migrated with synthetic 2'—O-methylcytidine in the n-butyl alcohol-concentrated ammonia-water (86:5:1#) system (15). In a separate experiment, a commercial sample of the nucleo- side cytidine was purified by a similar Dowex column and the percent methylation detected in the isolated cytidine was decreased from 0.15% to 0.05%, a virtual background level. Therefore, the values recorded in Table 2 represent true O-methyl content in these com- pounds, indicating that commercial samples of synthetic polynucleo- tides and component nucleosides and nucleotides contain measureable 2'-0-methyl groups, perhaps reflecting their biological origin. Also tested were the copolymers Poly (A,C) and Poly (C,U) which were synthesized with polynucleotide phosphorylase using the same nucleoside diphOSphates which had been assayed (Table 2). These polymers show evidence of incorporation of the 2'-0-methyl compounds prese incor than nucle 2’-1- acid, chrom tyros while O-met' Q-metl prese] tions and p( and p be see eXcel‘. made deter 10 present in the nucleoside diphOSphates. There seems to be greater incorporation of the 2'-0-methy1ated compounds in the Poly (C,U) than in the Poly (A,C), confirming previous experiments with poly- nucleotide phosphorylase and 2'-0-methylnucleotides (15). In order to determine whether O-methyl groups other than the 2'-0-methy1 group of ribose are released as methanol by perchloric acid, three other compounds were subjected to hydrolysis and gas chromatography. The amino acids DL-O-methylserine and L-O-methyl- tyrosine gave values of 59 and #5 mole percent O-methyl reSpectively while 5-0-methy1 glucose yielded a figure of 71 mole percent. These O-methylated compounds are not as completely hydrolyzed as pentose O-methyl groups under these conditions. Nevertheless, the possible presence of these and other O-methylated compounds in RNA prepara- tions make it imperative that the RNA be treated to remove proteins and polysaccharides as described in Materials and Methods. Synthetic 2'-0-Methylated Nucleosides and Polymers In Table 5 the results of determinations on synthetic monomers and polymers known to contain 2'-O-methyl groups are shown. As can be seen, the values for the synthetic monomers and polymers are in excellent agreement with determination of 2'-O-methylnucleotides made by paper chromatography over a wide range of 2'-0-methy1 content. Natural RNA Species In Table #, values are presented from the literature based on determinations of 2'-O-methylnucleotides in RNA obtained from a ll variety of organisms. The values range from bacterial ribosomal RNA, with 0.1% sugar methylation, to the wheat germ ribosomal RNA value of 1.7%. These data were obtained by various column chromato- graphic procedures all of which involved the use of large amounts of RNA. The values we have determined using the gas chromatographic method are also shown. It is apparent that good agreement is ob- tained between the two sets of data. In order to compare directly values determined by conventional column chromatographic techniques and those from gas chromatography, experiments were run in parallel using both methods to assay the 2'-0-methyl content of wheat germ rRNA and yeast tRNA. The amount of 2'-0-methylnucleotides was first determined by alkaline hydrolysis with l M NaOH at room temperature for 96 hours (5) followed by treatment with E, 221; alkaline phos— phatase. The nucleosides and dinucleoside monophosphates which resulted from this treatment were neutralized with 002 and diluted to a salt concentration of 0.01 M. The solution was applied to a DEAE-cellulose column in the formate form and the column was washed extensively with 0.01 M ammonium formate to remove all traces of the nucleosides. The dinucleoside monophosPhates were then eluted with 0.5 M ammonium formate. Absorbance at 260 nm was used to quantitate the yield of the alkali-stable dinucleoside monophosPhates and, thus, the mole-percent of sugar methylated nucleotides. Ali- quots of the same RNA sample were used for the gas chromatographic determination of 2'-0-methy1 content. The results presented in Table 5 Show that the values agree within the experimental errors of the techniques. 12 The effects of varying the amount of RNA in the samples used for determination of 2'-0-methy1 content have also been studied using yeast tRNA. Figure 2 Shows the results of such an experiment. As can be seen, at very high levels of RNA, there is a depression in the values recorded. This occurs because the assay conditions were kept the same over the whole range of concentrations and, in so doing, the liquid phase after centrifugation became smaller with large amounts of RNA present. If larger amounts of perchloric acid are used (e.g., #0 ul instead of 50 ul), the values obtained for 2'-0-methyl content at 800 ug are comparable with those using smaller amounts (250 ug) of RNA. When yeast tRNA samples of less than 150 ug are used, there is an apparent elevation of the 2'-0-methy1 content due to the contribution of water to the background noise, thus making the measurement of the small methanol peak difficult. If the RNA used is more highly methylated than the 0.9% of yeast tRNA, this lower threshold figure of 150 ug can be greatly reduced. DISCUSSION Ion exchange (5) and liquid partition (7) column methods for quantitating 2'-0-methyl content in RNA have several limitations. Hydrolysis of the RNA by either enzymes or alkali requires from 2# to 96 hours which must then be followed by column chromatographic separation for each sample. There is also the disadvantage that large amounts of RNA are necessary for determinations of high accu- racy. In addition, extraneous ultraviolet absorption by column materials and eluants can interfere with the measurement of 2'-0-methy1 13 content. If it is possible to label the methyl group by use of radioactive methyl-labeled methionine, the amount of sample used can be reduced and background interference is decreased. However, this technique requires $2.2122 conditions favorable for methyl- labeling and the long hydrolysis period is Still necessary, followed by chromatographic methods to distinguish base methylation from sugar methylation. The perchloric acid hydrolysis of 2'-0-methyl groups combined with gas chromatographic analysis of the released methanol provides an efficient method for measurement of 2'-0-methyl content in RNA. It does not require large amounts of sample and can be carried out rapidly. Because of the high resolution and sensitivity of the gas chromatographic technique, it is possible to measure methanol accurately in nanogram amounts. Thus, one can determine the 2'-0- methyl content of RNA samples from one-hundredth to one-thousandth the size necessary for determinations by conventional column methods. Radioactive labeling is not required so there are no restrictions on the Species of RNA which can be studied. In addition, the rapidity of the method makes it possible to process 15 samples in a day. The method has been shown to be accurate and reproducible in deter- minations of 2'-0-methy1 content in both synthetic and natural poly- nucleotides. There is no interference by DNA or by base methylated compounds. One stringent requirement for accuracy in the gas chromatographic method is that the sample be free of substances other than RNA which release methanol upon perchloric acid digestion. These substances appear to be primarily polysaccharides since they can be eliminated by treatment of the RNA as described in Materials 1# and Methods, a treatment which is an integral part of the method if impurities in the RNA are suSpected. SUMMARY A technique is described for the determination of 2'-0-methy1- ation in RNA using gas chromatography to quantitate the methanol released by perchloric acid cleavage of the 2'-methoxy group. The technique can be used with small samples of RNA and does not require radioactive labeling. It is shown that base methylated compounds do not interfere with the assay and that the method is accurate over widely ranging contents of 2'-0-methylnucleotides. ACKNOWLEDGMENT We wish to thank Dr. W. W. Wells for his guidance and assist- ance in setting up the gas chromatographic system. 2'-0-Methyl Determinations on Base Methylated 15 TABLE 1 Purine and Pyrimidine Compounds Compound Mole % Detected as 2'-0-Methyl Ne-Methyladenine Ne-Dimethyladenine N6-Isopentenyladenine l-Methylguanine 7-Methylguanine N2-Dimethylguanosine 5-Methylcytosine