Molec. Biol. Rep. Vol. 4, 2:105-110

ANALYSIS OF THE METHYLATED 'CAP' STRUCTURES OF VACCINIA mRNA BY TWO-DIMENSIONAL THIN-LAYER CHROMATOGRAPHY Hans J. GROSS t , Hans KROATH 2 , Hans-Georg JANDA 2 & Christoph JUNGWIRTH 2

1Max_Planck_institut fiir Biochemie, D-8033 Martinsried, IV. Germany 2Institut flit Virologie undlmmunbiologie der Universitdt Wi~rzburg, Versbacher Landstrasse 7, D-8700 h/i~rzburg, W. Germany (Received February 26, 1978)

Abstract Two different twodimensional cellulose thinlayer separations for blocked, methylated mRNA 5'-termini are described. They allow rapid analysis even of complex mixtures of mRNA "cap" structures on the basis of their methyl group content and base composition. These simple procedures are especially useful for the analysis of [3 H-methyl] -labeled mRNA in combination with tritium fluorography. A qualitative and quantitative analysis of the methylated "cap" structures of in vitro labeled Vaccinia "core" mRNA is presented. The presence of methylated "cap" structures in Vaccinia RNA increases the in vitro translation efficiency of methylated Vaccinia RNA over Vaccinia RNA transcribed in the absence of a methyl group donor.

Introduction In recent years it became evident that eukaryotic mRNAs of cellular and viral origin, with very few exceptions, carry blocked, methylated 5'-termini (mRNA "caps") of the general structure mTG(5')ppp(5')Xm or m7G(5')ppp(5')XmpYm (7, 15). As far as these RNAs were 32p-labeled, isolation and analysis of their 5'-terminal "cap" structures can easily be achieved by standard onedimensional electrophoresis (9) or twodimensional fingerprinting procedures (1, 7). Most frequently, however, mRNAs are labeled with [3 H-methyl] -menthionine in vivo or [3H-methyl]-S-adenosyl-methionine in vitro. The standard isolation process for such (3H)-labeled mRNA 5'-termini involves chromatography on DEAE-cellulose columns in the presence of urea

(reviewed in 7), which separates mRNA cap structures mainly according to their negative charge. This column chromatography is time consuming, the samples have to be desalted and only a very limited number of mRNAs can be analyzed at the same time. The twodimensional chromatographic systems used here allow the simultaneous separation of large numbers even of complex mixtures of mRNA 5'termini. We tested the usefulness of the twodimensional chromatography system on "early" Vaccinia mRNA transcribed in vitro in the presence of [3H-methyl]S-adenosylmethionine. Coupled with transcription, the "capping" and methylation of Vaccinia virus can be achieved in vitro by means of virion-associated enzymes (10, 18, 19). In a series of studies, the sequence of reactions leading to the two complete "cap I" structures mTGpppAm and m7GpppGm in Vaccinia mRNA has been clarified (11). The importance of the methylated "cap" is demonstrated by a comparison of the translation efficiencies of methylated and unmethylated Vaccinia core mRNA in a cell-free protein synthesizing system.

Materials and methods Precoated 20 x 20 cm cellulose thinlayer plates were bought from E. Merck, Darmstadt. Methylated mRNA termini (Fig. 1) and nucleosides (Fig. 3) were bought from PL-Biochemicals, Milwaukee. N6-MethyladenOsine(m6A), which occurs as "interhal" methylated nucleoside in mRNA (12, 17) was synthesized as described by Jones & Robins (4). Solvents for twodimensional thinlayer chromatography were solvent A: isobutyric acid-concentrated 105

ammonia-water, pH 3.7 (66:1:33; v:v:v); solvent B: saturated ammonium sulfate-1 M sodium acetate, pH 5.5 - propane-2-ol (40 9:1; v:v:v); solvent C: propane-2-ol - 1 N hydrochloric acid - ammonium chloride (25:25:1 ; v:v:w). The separation in the first dimension (solvent A) takes around 8 hrs. After drying the plates overnight under a hood, the second dimensions were developed in 6-7 hrs. in solvents B or C. Marker nucleotides and nucleosides were located on the thlnlayer plate with 254 nm UV light. [3Hmethyl]-labeled nucleotides were located by fluorography (13), whereby the thinlayer plate was exposed to X-ray film for 5-7 days at -70~ For further analysis, the methylated nucleotides were scraped from the plate, the scintillator was removed with ether and the radioactive material was eluted quantitatively from the cellulose powder with 1 ml water. [3H-methyl]-labeled Vaccinia mRNA was synthesized in the presence of 1 /aM [3H-methyl]-Sadenosylmethionine as described by Wei & Moss (18). Digestion with RNase T2 (Sankyo)was carried out for 4 - 5 hrs at 37~ in 30/al 5 mM potassium acetate pH 4.7 with two units of enzyme per A26o unit of RNA. For subsequent digestion with bacterial alkaline phosphatase (Boehringer), solutions were adjusted to pH 8.5 with 2 M triethylammoniumbicarbonate and incubated for 4 - 5 hrs. at 37 ~ in 100/al with 0.035 units of enzyme per A26o unit of RNA. The digests were dried, redissolved in an aqueous solution containing 9 unlabeled authentic "cap" markers as well as m6A, and subjected to chromatography. For further analysis of the "caps" eluted from the thinlayer plate, the 1 ml aqueous samples were lyophllized and redissolved in 50 /al water. To 25 /al of this solution, the following solutions were added: 10 /al snake venom phosphodiesterase (Boehringer, 1 mg/ml), 2 /al bacterial alkaline phosphatase (10 units/ml), 0.5/al 1 M triethylammoniumbicarbonate buffer pH 9.0, and 0.5 /al 100 mM MgCI2. After incubation at 37~ for 1.5 hrs, the material was dried in a desiccator overnight and redissolved in 4 /al of an aqueous marker solution containing 7 methylated nucleosides. Each 2 gl of this solution was subjected to thinlayer chromatography in solvents propane-2-ol-concentrated ammonia-water (70:10:20; v:v:v) and solvent A, respectively. The authentic marker nucleosides were located on the plate with 254 nm UV light. The [3 Hmethyl] -labeled nucleosides were located by scraping 0.5 cm strips of cellulose into counting vials using a semiautomatic thinlayer scraper (5) and subsequent 106

scintillation counting. For in vitro translation experiments methylated Vaccinia mRNA was transcribed with purified virions according to Kates & McAuslan (6) in the presence of 10/aM S-adenosylmethionine. Unmethylated Vaccinia mRNA was transcribed without S-adenosyl-methionine. The mRNAs were isolated as previously described (3). For characterization of the RNAs, samples were electrophoresed on 2.4% polyacrylamide gels containing 0.5% sodiumdodecylsulfate and 0.5% agarose as described by Weinberg et al. (20). In vitro translation was performed in a wheat germ system according to Roberts & Paterson (14). Optimal ionic concentrations for translation of methylated as well as unmethylated Vaccinia mRNA were 60 mM K § and 3.5 mM Mg2§ Newly synthesized polypeptides were labeled with 120 /aCi/ml [3H]leucine (Amersham Buchler, specific activity 35 Ci/ raM).

Results and discussion DEAE cellulose chromatography in 7 M urea separates mRNA 5'termini according to their negative charge. For the analysis of more complex mixtures of mRNA "caps" and of more samples at the same time, rapid and simple procedures are needed which, in addition to negative charge, separate such mRNA "cap" structures also according to their base composition and methyl group content. Figure 1 shows that the twodimensional thinlayer chromatography systems used here fulfil these requirements and that they can successfully be applied for the separation of complex mixtures of mRNA 5'-termini. For both separations (Fig. 1A and 1B), the same solvent is used in the first r~A

i~

B

A

I

rn?~ppp~m

m~Gppp ~,m mTGpppA

m'Gppp G ~

m-GpppGm 0

GpppGm

.~ (~--

m GpppUm

m~fppp m-~Am

C~O m" Gppp Cm GppP Am

m7GpppCm

GDop~m

STARI

I

9 " ~ppp m ~A m

r~ Gpp~

0 m~A

~ 0

m'GpppG (~ ~00\

0 m G OpD Um

m: Gppp G GpDO Gr~ ~ 2

SlJ~RI

~

]

Fig. 1. Twodimensional thinlayer chromatograms of mRNA 5'-termini. Nine commercially available "caps" as may be obtained after nuclease P, digestion of mRNA, and m6A were separated with solvent A in the first dimension, and solvent B (Figure 1A) and C (Figure 1B), respectively, in the second dimension. Approximately 0.05 A~,, units of each of the synthetic "cap" structures were subjected to chromatography and visualized under a 254 nm UV lamp.

dimension, whereas two different solvents are used for the second dimensions. It is evident that this separation o f " c a p " structures in both chromatograms occurs on the basis o f methyl group content and base composition. Simple mobility shifts are observed if methyl groups are added either to form mTG or a methylated ribose moiety. In case that elution o f m R N A "caps" from the plates is needed for further characterization, solvent C should be applied for the second dimension (Fig. 1B), since the ammonium sulfate in solvent B (Fig. 1A) creates a desalting problem. [3 H.methyl] .labeled mRNA "cap" structures, located on the plate by fluorography (13), can easily and quantitatively be eluted in the way described above. Overnight evaporation over phosphopentoxide and potassium hydroxide removes the ammonium chloride from the sample. The successful application o f this twodimensional separation for the isolation and characterization o f 5'-termini from [3H-methyl]-Vaccinia core m R N A is shown in Fig. 2. In these experiments, however, we prefered digestion o f the in vitro labeled tran-

script (18) with RNase T2 and bacterial alkaline phosphatase. This results in m R N A "caps" o f the general structure mTGpppXmpY with an unmethylated nucleoside in 3' position, in contrast to "caps" o f the general structure mTGpppXm obtained after nuclease P1 treatment a's shown in Fig. 1. Figures 1 and 2 demonstrate that even complex mixtures of both types of " c a p " structures are well separated. Depending on the Vaccinia core mRNA preparations, up to 9 [3 H-methyl] -labeled "cap" structures can be detected after twodimensional chromatography and subsequent fluorography (Fig. 2, left part). For identification o f the [3H-methyl]labeled "caps", the radioactive material corresponding to spots number 1 - 7 was recovered from the plate and further analyzed by enzymic digestion with snake venom phosphodiesterase and bacterial alkaline phosphatase. This treatment cleaves the 5' - 5' triphosphate bridge and renders methylated mononucleosides which then can be identified by chromatography as described in Materials and methods. Figure 3 shows the identification of the "cap I"

Fig. 2. Twodimensional thinlayer chromatogram of the [3 H-methyl]-labeled 5':termini of in vitro transcribed Vaccinia mRNA. Approximately 5 x 104 cpm of the RNase T~ and bacterial alkaline phosphatase digest was subjected to chromatography using solvent A in the first dimension and solvent C in the second dimension. The added optical density markers as well as nucleosides derived from carrier tRNA were visualized under 254 nm UV light. Radioactive spots were located by fluorography according to Randerath (13) and identified as described in Materials and methods. The positions of the optical density markers are outlined solid, positions of radioactive nucleotides are indicated by arrows and numbered (Figure 2, left part) or outlined broken (Figure 2, right part). The identified radioactive "cap" structures are listed in the right part of Figure 2. The film was exposed for 4 days (fluorography at -70~ Spots 1 and 2 turned out to be identical due to streaking of mTGpppG. 107

"

GO0 9

9

,~176 t

200 9

u Z

100

"

t~

to Distance

20

[cm]

150

B

100 9

E CL u

S0-

2O Distance

[cm]

Fig. 3. Identification of a "cap I" structure derived from Vaccinia mRNA transcribed in vitro in the presence of [3Hmethyll-S-adenosylrnethionine. The radioactive material of spot number 6 in Figure 2 was recovered from the plate and digested with snake venom phosphodiesterase and bacterial alkaline phosphatase as described in Materials and methods. Solvents for chromatography were propane-2-ol-concentrated ammonia-water (70:10:20; v:v:v) (Figure 3A) and solvent A (Figure 3B). The positions of the added optical markers visualized with 254 nm UV light are outlined solid. The [3H-methyl]labeled nucleosides were located by scraping 0.5 cm strips from the plate and subsequent liquid scintillation counting of each fraction. structure derived from radioactive spot number 6 o f Figure 2. The radioactive material was recovered from the plate and digested with snake venom phosphodiesterase and bacterial alkaline phosphatase. One half o f the enzyme digest was chromatographed with solvent propane-2-ol-concentrated ammoniawater (70:10:20; v:v:v), the other half was developed in solvent A. 0.5 cm strips of the cellulose were then scraped off the plate and the radioactive material in each fraction was assayed by liquid scintillation 108

counting. By comparison o f the pattern o f radioactivity with the corresponding position o f the added authentic nucleosides visible in 254 nm UV light, a very accurate identification of the [3Hmethyl]-labeled nucleosides is achieved. In this way, all the radioactive mRNA "caps" corresponding to spots number 1 - 7 in Figure 2 were identified (see Fig. 2, right part). In agreement with results reported by Moss et at. (10), the two predominant "cap I" structures mTGpppGm and mTGpppAm as well as their incomplete counterparts mTGpppG and mTGpppA were thus detected in in vitro transcribed Vaccinia core mRNA. The results o f "cap" analyses o f three different samples of Vaccinia core m R N A synthesized in the presence of 1 tzM [3H-methyl]S-adenosylmethionine are summarized in Table 1: between 30 - 50% o f the radioactivity is incorporated into roughly equal amounts of m7 GpppGmpN and mTGpppAmpN, the two predominant "cap I" structures in Vaccinia mRNA. The high amount of "incomplete cap" structures o f the type mTGpppG or mTGpppA might be the result o f the limiting concentration o f the methyl donor present during in vitro transcription (10). It is o f interest to note the unequal distribution o f radioactivity incorporated into mTGpppG and mTGpppA, respectively. The significantly higher amount o f G compared to A in the penultimate position is different from the situation with Vaccinia mRNA isolated from infected chick cells (8). Surprisingly, we detected, in one out of three samples, a small amount of mTGpppm~Am (Table 1), a "cap" which has so far been described only Table 1. Total amount of methylated 5'-termini of three different samples of Vaccinia mRNA transcribed in vitro in the presence of 1 uM [3H-methyl]-S-adenosylmethionine. Methylated "cap" structures were analyzed by twodimensional chromatography like the one shown in Figure 2. From the radioactive nucleotides recovered quantitatively from the plates, aliquots from each "cap" were measured by liquid scintillation counting. All numbers represent percentage of [3H-methyl]-cpm of total [3H-methyl]cpm recovered from the corresponding chromatogram. methylated 5'-terminus mTGpppGmpN m 7GpppAmpN m 7Gpppm 6 AmpN mTGpppG mTGpppA m 7GpppN (total)

experiment number 1

2

3

18 14

16 16.5 2 44 20

28 22 45 5

(98.5)

(100)

44 17 4 (97)

being present in viral mRNA isolated from infected cells (2). We believe this to be due to the higher sensitivity o f the detection system used here compared to the system applied so far for "cap" analysis o f Vaccinia mRNA. In order to substantiate the necessity of the methylated 5'-termini for efficient translation of Vaccinia mRNA, we investigated the response of a wheat germ protein synthesizing system to added methylated compared to unmethylated in vitro transcribed Vaccinia mRNA. The electrophoretic mobility pattern of Vaccinia core mRNA transcribed 18S

n 28S

in the presence or absence of 10 /aM S-adenosylmethionine are virtually identical (Fig. 4). Under optimal ionic concentrations (which were 60 mM K § and 3.5 mM Mg2§ both for methylated and unmethylated Vaccinia mRNA), the reaction was saturated with 70 to 90/ag/ml o f each RNA (Fig. 5).

II 15

o

o

x t z

m[~NA

s s~

,A

IA

=i,

i,,I/,',,, Fig. 5. Incorporation of ['Hi-leucine into acid insoluble material in the wheat germ system stimulated by increasing amounts of added methylated in comparison to unmethylated Vaccinia mRNA. The reaction was carried out under optimal ionic concentrations as described in Materials and methods. o--~, addition of methylated Vaccinia mRNA (Vac(MET)mRNA) o--u, addition of unmethylated Vaccinia mRNA (Va.cmRNA). The kinetics of protein synthesis in response to methylated and unmethylated Vaccinia mRNA is shown in Figure 6. Incorporation of [a H] -leucine into acid insoluble material occurred for about 90 minutes and then leveled off in both cases. However, methylated Vaccinia mRNA stimulated protein synthesis at least twice as efficiently as unmethylated Vaccinia mRNA added in equal amounts.

Vac mRNA

Vac MET mRNA

8'0

6'0

4'o

2'o

0

Fractions Fig. 4. SDS-polyacrylamide gel analysis of Vaccinia mRNAs transcribed in vitro in the presence or absence of S-adenosylmethionine. Samples of 80 ~g of methylated or unmethylated Vaccinia RNA were subjected to electrophoresis on 2.4% polyacryiamide-agarose gels as described in Materials and methods. 18S and 28S rRNA were added as markers. Optical density was measured at 260 nm in a Zeiss gel scanner.

A detailed comparison of the in vitro synthesized products directed by Vaccinia core RNA and viral proteins isolated from infected cells is presently under investigation. Since it is known that wheat germ extracts contain enzymes catalyzing the methylation of added unmethylated mRNAs, we investigated the influence of S-adenosylhomocysteine, a potent inhibitor of methylation, on the translation efficiencies of methylated and unmethylated Vaccinia mRNA (Table 2). Whereas translation of methylated Vaccinia 109

H-

10-

!i-

m-

II

//

__

S z

from methylation during in vitro translation in the cell free system. Addition o f the methyl donor S-adenosylmethionine did not have any influence on translation efficiency. Taken together, these results suggest a decisive role o f methylation for the in vitro translation efficiency of Vaccinia core RNA, similar to the situation with other viral mRNAs (15). A recent report by Weber et al. on the functional efficiency o f methylated and unmethylated Vaccinia core RN A leads to similar conclusions (16). We believe that the very precise and sensitive method for m R N A " c a p " analysis presented here will contribute towards clarifying the biological role o f these unusual structures.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (Ju 76/11). Fig. 6. Time course of [3H] 4eucine incorporation into acid insoluble material in the wheat germ cell-free system stimulated by methylated and unmethylated Vaccinia-mRNA. Virus mRNAs and reaction mixtures were prepared as described in Materials and methods. 10 tJlaliquotswere removed at indicated intervals and precipitated in 5% TCA. ~, addition of methylated Vaccinia mRNA (Vac(MET)mRNA) u--o, addition of unmethylated Vaccinia mRNA (VacmRNA). u--u, without added mRNA (endogenous).

Table 2. Influence of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) on in vitro translation of methylated and unmethylated Vaccinia mRNA. The reaction mixtures were incubated for 2 hrs at 30~ under standard conditions (see Materials and methods). 5 t~l aliquots were used to measure the acid insoluble radioactivity. Where indicated, SAM (4 zM) and/or SAH (320 zM) were added to the reaction mixture. Numbers represent [ 3H ] -leucine incorporation. mRNA

amino acid incorporation (clam) -SAM + S A M + S A H +SAM -SAH +SAH

Vac(Met)mRNA Vac mRNA endogenous

2029 1104 322

2184 1227 274

2587 859 263

2228 856 300

m R N A was not hampered by addition of the inhibitor, the translation efficiency o f added unmethylated Vaccinia m R N A was reduced by 30 - 50%. This means that at least part o f the translation efficiency o f originally unmethylated Vaccinia m R N A stems

! 10

References 1. Adams, J.M. & S. Cory. Nature 255:28 (1975). 2. Boone, R.F. & B. Moss. Virology 79:67 (1977). 3. Jaureguiberry, G., F. Ben-Hamida, F. Chapeville & G. Beaud. J. Virol. 15:1467 (1975). 4. Jones, J.W. & R.K. Robins. J. Am. Chem. Soc. 85: 193 (1963). 5. Kasang, G., G. G61dner & N. Weis. J. Chromatography 59:393 (1971). 6. Kates, J.R. & B.R. McAuslan. Proc. Natl. Acad. Sci. USA 58:134 (1967). 7. Klootwijk, J., J. Klein, P. Zabel & A. van Kammen. Cell 11:73 (1977). 8. Kroath, H., H.-G. Janda, G. Hiller, E. Kuhn, C. J ungwirth, H.J. Gross & G. Bodo (subm. for publ.). 9. Mory, Y.Y. & M.L. Getter. Nucleic Acids Res. 4:1739 (1977). 10. Moss, B., A. Gershowitz, C.-M. Wei & R.F. Boone. Virology 72:341 (1976). 11. Moss, B., S.A. Martin, M.J. Ensinger, R.F. Boone & C.-M. Wei. pp. 63-81, in: W.E. Cohn & E. Volkin (eds.), Progress in Nucleic Acid Research and Molecular Biology, Vol. 19. Academic Press, New York (1976). 12. Perry, R.P. & D.E. Kelley. Cell 8:433 (1976). 13. Randerath, E., C.-T. Yu & K. Randerath. Analyt. Biochem. 48:172 (1972). 14. Roberts, B.E. & B.M. Paterson. Proc. Natl. Acad. Sci. USA 70; 2330 (1973). 15. Shatkin, A.J.CeI~ 9:645 (1976). 16. Weber, L.A., E.D. Hickey, D.L. Nuss & C. Baglioni. Proc. Natl. Acad. Sci. USA 74:3254 (1977). 17. Wei, C.-M., A. Gershowitz & B. Moss. Biochemistry 15:397 (1976). 18. Wei, C.-M. & B. Moss. Proc. Natl. Acad. Sci. USA 71: 3014 (1974). 19. Wei, C.-M. & B. Moss. Proc. Natl. Acad. Sci. USA 72: 318(1975). 20. Weinberg, R.A., U. Loening, M. Willems & S. Penman. Proc. Natl. Acad. Sci. USA 58:1088 (1967).