5
Eur J. Biochem. 134, 117-121 (1983) I( FEBS 1983 Partial Purification and Characterization of mRNA (Guanine-7-)Methyltransferase from the Yeast Saccharomyces cerevisiae Camille LOCHT, Jean-Luc BEAUDART, and Jean DELCOUR Unite de Genetique Moleculaire des Eucaryotes, Universite Catholique de Louvam, Louvain-la-Neuve ; and Laboratoire de Genetique Moleculaire et de Physiologie Cellulaire, Facultes Universitaires Notre-Dame de la Paix, Namur (Received April 6, 1983) - EJB 83 0345 As a tool for the study of the capping-methylation process of yeast mRNA, we developed a procedure for the purification of the mRNA (guanine-7-)methyltransferase using the commercial cap analog guano- sine(5’)triphospho(5’)guanosine as a substrate and radioactive S-adenosylmethionine (AdoMet) as the methyl group donor. The osmotic-sensitive yeast strain VY 1160 was used as the enzyme source. Little methyltransferase activity was detectable in a crude lysate obtained after osmotic shock. We showed that this was due to the presence of a low- molecular-weight inhibitor which could easily be eliminated by Sephadex G-25 gel filtration. The 10000 x g supernatant from the crude lysate was submitted to DEAE-cellulose and DNA-agarose chromatography. The resulting preparation was enriched about 450-fold in specific activity. Under standard assay conditions, the incorporation rate remained constant for at least 6 h at 30 “C. Transmethylation was not stimulated by KCI nor NaCI. Divalent cations were strong inhibitors. The partially purified enzyme was able to methylate undermethylated poly(A)-rich mRNA isolated from an AdoMet auxotrophic yeast strain briefly exposed to AdoMet-free medium. Methylated 5’-terminal cap structures have been described in most eukaryotic and many viral mRNAs. In these structures an N7-methylguanosine is attached through a 5‘-5‘ triphosphate bridge to the penultimate nucleoside (cap 0). In some molecules additional 2‘-O-ribose methylations are found at the penul- timate (cap 1) and the antepenultimate nucleotides (cap 2) (for a review, see Banerjee [I]). The complexity of the cap increases as one moves up the evolutionary scale from yeast (cap 0) [2, 31 to mammals (cap 1 and 2) [4]. The microbial eukaryote Saccharomyces cerevisiae is an ideal organism for probing the enzyme mechanisms of mRNA capping and methylation. It offers the advantage over higher cells of rapid growth and relatively simple organization (cap 0 structure). In addition, yeast cells can exist stably in both haploid and diploid states, thereby facilitating the isolation of mutants and their genetic analysis. A combined genetic and biochemical approach for dissecting out the capping and methylation machinery should therefore be feasible in yeast. In this communication we report the partial purification and characterization of the mRNA (guanine-7-)meth- yltransferase from the osmotic-sensitive strain VY 1160 [5]. In our procedure G5pppsG serves as the acceptor molecule and [Me-3H]AdoMet as the methyl donor to produce radioactive m7G5‘pppsG caps. The enzymatic reaction is inhibited by AdoHcy. The purified enzyme fraction is shown to methylate undermethylated yeast poly(A)-rich mRNA extracted from an AdoMet auxotrophic mutant strain briefly exposed to an AdoMet-free medium. MATERIALS AND METHODS Materials [Me-3H]AdoMet was purchased from Amersham Inter- national. GspppsG, m7G5’ppp5’G, m7G5’ppp5’A, PI nuclease and DNA-agarose were obtained from P-L Biochemicals. RNase T2 and AdoHcy were supplied by Sigma. DEAE- cellulose (DE52), DEAE-cellulose filters (DESI, 2.3 cm diam- eter), Whatman 3 MM chromatography paper and What- man GFjC glass fiber discs came from Whatman. Pharmacia Fine Chemicals was the source of Sephadex G-25. Cell Cultures The osmotic-sensitive strain VY 1 160 of Saccharornyces cerevisiae was kindly supplied by Dr P. Venkov (Bulgarian Academy of Sciences, Molecular Biology Laboratory, Sofia, Bulgaria). Cells were grown in suspension culture in 1 1 YM-5 medium supplemented with 10 % sorbitol at 30 “C [5]. The AdoMet-dependent strain CC30-11 D (sam 1-11, Sam 2-1 2), kindly provided by Y. Surdin-Kejan (Laboratoire d’Enzymologie du CNRS, Gif-sur-Yvette, France), was grown in 1 1 minimal medium supplemented with AdoMet [6] at 30 “C to a density of 0.4 A420 unit and then shifted to fresh medium lacking AdoMet. Incubation was continued for 60 min and the cells were harvested to prepare undermethylated poly(A)-rich mRNA. Abbreviations. AdoMet, S-adenosyl-methionine; AdoHcy, S-aden- osyl-homocysteine; GS’pppS’G, guanosine(5’)triphospho(5’)guanosine; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonk acid; buffer A, 100 mM Tris/HCl, pH 9.0,l mM EDTA, 100 mM NaCl; buffer B, 50 mM Tris/HCl, pH 7.9, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 Triton X- 100, 25% ethylene glycol; NaAc, sodium acetate. Enzymes. mRNA (guanine-7-)methyltransferdse (EC 2.1 ,l. -): RNase T2 (EC 3.1.27.1); RNase PI (EC 3.1.30.1). Preparation of’ Undermethylated Poly (A)- Rich mRNA 1 g of cells were suspended in 2.5 ml buffer A. 2.5 ml buf- fer-A-saturated phenol and 1.5 g of glass beads (diameter 0.45 mm) were added. The cooled suspension was shaken for 60 sin a Braun cell homogenizer (MSK) and then centrifuged at 1000 x g for 15 min. The aqueous phase was reextracted three times with phenol and the total RNA was precipitated over-

Partial Purification and Characterization of mRNA (Guanine-7-) Methyltransferase from the Yeast Saccharomyces cerevisiae

Embed Size (px)

Citation preview

Page 1: Partial Purification and Characterization of mRNA (Guanine-7-) Methyltransferase from the Yeast Saccharomyces cerevisiae

Eur J. Biochem. 134, 117-121 (1983) I( FEBS 1983

Partial Purification and Characterization of mRNA (Guanine-7-)Methyltransferase from the Yeast Saccharomyces cerevisiae

Camille LOCHT, Jean-Luc BEAUDART, and Jean DELCOUR

Unite de Genetique Moleculaire des Eucaryotes, Universite Catholique de Louvam, Louvain-la-Neuve ; and Laboratoire de Genetique Moleculaire et de Physiologie Cellulaire, Facultes Universitaires Notre-Dame de la Paix, Namur

(Received April 6, 1983) - EJB 83 0345

As a tool for the study of the capping-methylation process of yeast mRNA, we developed a procedure for the purification of the mRNA (guanine-7-)methyltransferase using the commercial cap analog guano- sine(5’)triphospho(5’)guanosine as a substrate and radioactive S-adenosylmethionine (AdoMet) as the methyl group donor. The osmotic-sensitive yeast strain VY 1160 was used as the enzyme source. Little methyltransferase activity was detectable in a crude lysate obtained after osmotic shock. We showed that this was due to the presence of a low- molecular-weight inhibitor which could easily be eliminated by Sephadex G-25 gel filtration. The 10000 x g supernatant from the crude lysate was submitted to DEAE-cellulose and DNA-agarose chromatography. The resulting preparation was enriched about 450-fold in specific activity. Under standard assay conditions, the incorporation rate remained constant for at least 6 h at 30 “C. Transmethylation was not stimulated by KCI nor NaCI. Divalent cations were strong inhibitors. The partially purified enzyme was able to methylate undermethylated poly(A)-rich mRNA isolated from an AdoMet auxotrophic yeast strain briefly exposed to AdoMet-free medium.

Methylated 5’-terminal cap structures have been described in most eukaryotic and many viral mRNAs. In these structures an N7-methylguanosine is attached through a 5‘-5‘ triphosphate bridge to the penultimate nucleoside (cap 0). In some molecules additional 2‘-O-ribose methylations are found at the penul- timate (cap 1) and the antepenultimate nucleotides (cap 2 ) (for a review, see Banerjee [I]). The complexity of the cap increases as one moves up the evolutionary scale from yeast (cap 0) [2, 31 to mammals (cap 1 and 2) [4].

The microbial eukaryote Saccharomyces cerevisiae is an ideal organism for probing the enzyme mechanisms of mRNA capping and methylation. It offers the advantage over higher cells of rapid growth and relatively simple organization (cap 0 structure). In addition, yeast cells can exist stably in both haploid and diploid states, thereby facilitating the isolation of mutants and their genetic analysis. A combined genetic and biochemical approach for dissecting out the capping and methylation machinery should therefore be feasible in yeast.

In this communication we report the partial purification and characterization of the mRNA (guanine-7-)meth- yltransferase from the osmotic-sensitive strain VY 1160 [5]. In our procedure G5pppsG serves as the acceptor molecule and [Me-3H]AdoMet as the methyl donor to produce radioactive m7G5‘pppsG caps. The enzymatic reaction is inhibited by AdoHcy. The purified enzyme fraction is shown to methylate undermethylated yeast poly(A)-rich mRNA extracted from an AdoMet auxotrophic mutant strain briefly exposed to an AdoMet-free medium.

MATERIALS AND METHODS

Materials

[Me-3H]AdoMet was purchased from Amersham Inter- national. GspppsG, m7G5’ppp5’G, m7G5’ppp5’A, PI nuclease and DNA-agarose were obtained from P-L Biochemicals. RNase T2 and AdoHcy were supplied by Sigma. DEAE- cellulose (DE52), DEAE-cellulose filters (DESI, 2.3 cm diam- eter), Whatman 3 M M chromatography paper and What- man GFjC glass fiber discs came from Whatman. Pharmacia Fine Chemicals was the source of Sephadex G-25.

Cell Cultures

The osmotic-sensitive strain VY 1 160 of Saccharornyces cerevisiae was kindly supplied by Dr P. Venkov (Bulgarian Academy of Sciences, Molecular Biology Laboratory, Sofia, Bulgaria). Cells were grown in suspension culture in 1 1 YM-5 medium supplemented with 10 % sorbitol at 30 “C [5].

The AdoMet-dependent strain CC30-11 D (sam 1-11, Sam 2-1 2) , kindly provided by Y. Surdin-Kejan (Laboratoire d’Enzymologie du CNRS, Gif-sur-Yvette, France), was grown in 1 1 minimal medium supplemented with AdoMet [6] at 30 “C to a density of 0.4 A420 unit and then shifted to fresh medium lacking AdoMet. Incubation was continued for 60 min and the cells were harvested to prepare undermethylated poly(A)-rich mRNA.

Abbreviations. AdoMet, S-adenosyl-methionine; AdoHcy, S-aden- osyl-homocysteine; GS’pppS’G, guanosine(5’)triphospho(5’)guanosine; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonk acid; buffer A, 100 mM Tris/HCl, pH 9.0,l mM EDTA, 100 mM NaCl; buffer B, 50 mM Tris/HCl, pH 7.9, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 Triton X- 100, 25% ethylene glycol; NaAc, sodium acetate.

Enzymes. mRNA (guanine-7-)methyltransferdse (EC 2.1 , l . -): RNase T2 (EC 3.1.27.1); RNase PI (EC 3.1.30.1).

Preparation of’ Undermethylated Poly ( A ) - Rich mRNA

1 g of cells were suspended in 2.5 ml buffer A. 2.5 ml buf- fer-A-saturated phenol and 1.5 g of glass beads (diameter 0.45 mm) were added. The cooled suspension was shaken for 60 sin a Braun cell homogenizer (MSK) and then centrifuged at 1000 x g for 15 min. The aqueous phase was reextracted three times with phenol and the total RNA was precipitated over-

Page 2: Partial Purification and Characterization of mRNA (Guanine-7-) Methyltransferase from the Yeast Saccharomyces cerevisiae

118

night with 2.5 vol. ethanol and 0.1 vol. NaAc (pH 5.5) at - 20 “C. The precipitated RNA was washed once with ethanol, twice with anhydrous ether and dried under an air stream. RNA was then dissolved in 10 mM Tris/HCl (pH 7.5) and poly(A)-rich mRNA was extracted by oligo(dT)-cellulose chromatography [2].

Protein Determinations

Protein concentrations was determinated by the Bradford method [7] using bovine serum albumin as a standard.

Electrophoresis

Conditions were essentially as described in Laemmli [8], except that acrylamide concentration in the running gels was 13 ”/,.

Methyltransferase Assay

The standard cap methylation reaction mixture (7.5 pl) contained 20 mM Hepes/KOH (pH 7.6), 1 mM dithiothreitol, 0.4 mM GS’ppp5’G, 0.2 pCi [Me-3H]AdoMet (15 Ci/mmol) and 2.5 1.11 of enzyme fraction, unless otherwise indicated. After 60 min (or 2 h when indicated) at 30 “C, 5 pl were spotted onto DEAE-cellulose filter paper discs and washed five times with 25 mM ammonium formate (pH 8.0), once with water and once withethanol. Dried filters were soaked in 0.5 ml of 1 M LiCl to elute the bound nucleotides, and counted in a toluene-based scintillation fluid containing 33 ”/, Triton X-100. When poly(A)-rich mRNA was methylated, the reaction mixture (50 pl) contained 20 mM Hepes/KOH (pH 7.6), 1 mM di- thiothreitol, 0.7 [jH]AdoMet (15 Ci/mmol), 10 pgpoly(A)-rich mRNA and 15 p1 ofenzyme extract. After 60 min of incubation at 30 “C the reaction was stopped with 0.5 ml of buffer A and 0.5 ml redistilled, buffer-A-saturated phenol. The mixture was shaken, centrifuged, and the phenol phase was reextracted with 0.5 ml buffer A. Both aqueous phases were combined and the RNA was precipitated by 10 ml cold 10% CCI,C02H, re- covered on 2.3-cm glass-fiber filters, washed with 20 ml cold 5 ”/, CCl,CO,H and 10 ml ethanol. The filters were dried and counted in a toluene-based scintillation fluid.

Analysis of the Methylated Nucleotides

To characterize the methylated RNA products further, the initial reaction mixture was scaled up fivefold (250 pl) and after a I-h incubation at 30°C the phenol-extracted RNA was precipitated with 2.5 vol. ethanol and 0.1 vol. 3 M NaAc (pH 5.5) overnight at - 20 “C, recovered by centrifugation, washed with ethanol and anhydrous ether, dried, resuspended in 0.5 ml 10 mM NaAc (pH 4.5) and incubated with 10 units RNase T,. After 17 h at 37 “C the pH was adjusted to 6.0 with NaOH, 20 units of RNase PI were added and the incubation was continued for 2 h. The treated RNA, as well as methylated cap analogs, were then analyzed by descending paper chroma- tography on Whatman 3 MM paper with isobutyric acid/0.5 M NH,OH (10/6, v/v). Authentic markers were visualized with ultraviolet light at 254 nm. Paper was cut into 1 x 2-cm sections and counted for radioactivity.

Purification Procedure

VY 1160 yeast cells were grown to 0.7 A,, , unit, harvested by centrifugation, washed twice with lox sorbitol and re-

Table 1. Incorporcrrion of [3HJrnrthyl groups hy yrast cell fractions Each reaction mixture (7.5 ml) contained 20 mM HepesiKOH (pH 7.6), 1 mM dithiothreitol, 0.2 pCi [Me-3H]AdoMet (15 Ci/mmol) and 2.5 pl of the different cell fractions. After 2 h at 30 ‘ C the methylated products were recovered as described in Materials and Methods

Fraction

~ ~~

[3H]Methyl incorporated _ _ ~ _ _ ~ - + GS ppp5 G ~ G’ ppp’ G (0.4 mM)

pmol -~~~~

1. Crude extract 0.56 0.47 2. Fraction not retained

by Sephadex G-25 1.02 0.52 3. Fraction retained by

Sephadex G-25 0.07 0.06 4. Fractions 2+3 0.28 0.22

suspended in 2 vol. water. All further steps were carried out at 0-4°C. The cells were disrupted with 15 strokes of a Potter- Elvehjem homogenizer. The lysed suspension was adjusted to buffer B and centrifuged at 10000 x g for 15 min. The super- natant was applied onto a DEAE-cellulose column (10 x 2 cm) equilibrated with buffer B and washed with 200 ml of the same buffer. Protein concentration was determined and enzyme activity was measured. Active fractions were pooled, diluted fourfold with buffer B, applied to a DNA-agarose column (0.8 x 4.5 cm) equilibrated with the same buffer. The column was

washed with 100 ml of buffer B and the bound material was eluted at flow rate of 12 ml/h with 50 ml of buffer B containing 0.3 M NaC1. Fractions containing the methyltransferase ac- tivity were combined and stored at - 80 ”C until further use.

RESULTS

Detection of m R N A (Guanine- 7 - ) methyltransferase Activity in the Yeast Lysate

As previously shown in the case of RNA methyltransferases isolated from other sources [9 - 121, the commercial G5pppS’G cap analog can be used as a specific acceptor for the methyl- ation at the N-7 position of at least one of the two guanosines. This dinucleotide was therefore incubated in the standard reaction mixture with the crude 10000 x g supernatant from the yeast cell lysate. Table 1 shows that the cap methyltransferase activity of this crude extract was very low indeed, since the amount of radioactivity which could be recovered on DEAE- cellulose filters was about the same whether or not cap analogs had been added to the sample. The cap-dependent activity could be significantly increased by using the fraction of the total crude extract eluted from Sephadex (3-25. The fraction bound to the gel did not show any activity but inhibited the reaction when recombined with the active fraction. This inhibitor also prevented the transfer of methyl groups to cap analogs in wheat germ extracts as well as to proteins in wheat germ and Escherichia coli extracts (results not shown), suggest- ing that we were dealing with some general inhibitor of transmethylation. Dialysis was not practicable since the en- zyme was rapidly destroyed in the crude lysate.

Page 3: Partial Purification and Characterization of mRNA (Guanine-7-) Methyltransferase from the Yeast Saccharomyces cerevisiae

119

Table 2. Summary of the purification of yea.rr m R N A jguanine-7-)meth~ltrans~er.u.se Transmethylation was assayed under standard conditions. Protein concentrations were determined by the Bradford method [7]

Fraction Volume Protein Activity Specific Purification Yield activity

ml mg pmol h- ' pmol h- ' -fold % (mg protein)- '

Crude extract 2.5 309.25 88 0.285 1 100 DE AE-cellulose 3 26.4 552 10.9 73 627 DNA-agarose 0.45 1.66 233 140.7 49 3 266

"0 5 10 15 Fraction number

Fig. 1. Purificution of m R N A (guanine-7-jmethyllransferase. Chroma- tography of methyltransferase on DEAE-cellulose (A) and DNA-agarose (B). Elution was performed using 0.3 M NaCl at a flow rate of 60 ml/h (A) and 12 ml/h (B), starting at the position indicated by an arrow. Protein concentration (0-0) and enzyme activity (0-0) were measured as indicated in Materials and Methods, the latter being given as amount of methyl group incorporated

Purifiicatio~ of the mRNA (Guanine-7-)methyltransfrrase

The purification scheme is summarized in Table 2. To limit the loss ofenzyme activity due to its instability at the unpurified stage, phase partition and high-speed centrifugation, as used in HeLa cell [lo] and wheat germ [12], respectively, were omitted. For the same reason dialysis and Sephadex (3-25 filtration were also omitted. The 10000 x g supernatant from the crude lysate was applied immediately onto a DEAE-cellulose column and the methyltransferase was eluted as a single peak by a 0.3 M NaCl batch elution (Fig. 1 A). This single step increased the specific activity about 70-fold and more than 600% of the activity was recovered. This obviously resulted from the removal of the inhibitor described in the previous section. After passage through the DEAE-cellulose column, incorporation became strongly dependent upon the addition of cap analog. Fractions 10 - 12 were combined, adjusted to the final 75 mM NaCl concentration and applied onto the DNA-agarose col- umn (Fig. IB). About 40% of the activity applied to this column was recovered, which gave a final purification of 490- fold relative to the crude extract activity. The actual degree of

A B C

Fig. 2. Analysis of'the purified methyltransferase,fraction by sodium dodecyl suljate/polyacrylarnide gel electrophoresis. The crude fraction (A) and the purified methyhdnsferase fraction (B) were analysed by electrophoresis on a polyacrylamide gel. The M, standards (C) are: phosphorylase b (94000), bovine serum albumine (68000), ovalbumin (43 000), carbonic anhydrase (30000), soybean trypsin inhibitor (21 000) and lysozyme (14300)

purification was probably higher since the enzyme was unstable during the initial steps of the procedure. Analysis of the final purified fraction by polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed clearly one single major band with an apparent molecular weight of 49000 (Fig. 2) which was similar to the molecular weight of the HeLa cell methyltrans- €erase (56000) [lo]. The purified enzyme could be stored at - 80 'C without any detectable loss of activity for at least several months.

Charucteristics of the Methyltransferuse Reaction

Under standard reaction conditions, incorporation of methyl groups into G5 ppp5 G by the purified enzyme continued at a linear rate for at least 6 h (Fig. 3). The temperature optimum was around 30 "C (Table 3A). Using several buffers, optimal methyltransferase activity was observed at pH 7.0 (Table 3 B). The half-maximum activity was at pH 5.0. Because AdoMet is very unstable in the alkaline range [I 31, the enzyme activity was not tested above pH 8.0. The influence of AdoMet concentration on the methyltransferase activity was measured

Page 4: Partial Purification and Characterization of mRNA (Guanine-7-) Methyltransferase from the Yeast Saccharomyces cerevisiae

120

Table 3. Effects of temperature und p H on the mRNA (guuriine-7-)- merhyltrunsferuse activity (A) Assays were conducted under standard reaction conditions at the different temperatures. (B) The effect of pH was measured at 30 C by replacing the 20 mM Hepes (pH 7.5) buffer by acetate (a), phosphate (b) or Hepes (c) buffers (each at 40 mM)

A. Temperature Me incorporated

“C 6

20 25 30 35 37

pmol 0.24 0.72 0.82 0.90 0.63 0.56

B. pH (buffer) M e incorporated

4.0 (a) 0.07 4.5 (a) 0.08 5.0 (a) 0.28 5.5 (a) 0.37 5.5 (b) 0.45 6 0 (b) 0.54 6.5 (b) 0.68 7.0 (b) 0.76 7.0 (c) 0.79 7.5 (b) 0.74 7.5 (c) 0.76 8.0 (c) 0.63

by changing the concentration of AdoMet while holding G5pppsG at a fixed concentration. An apparent K,,, of 2 pM was extrapolated from Lineweaver-Burk plots of the data. AdoHcy was found to inhibit strongly the incorporation of radioactivity. With 1 pM AdoHcy in the standard reaction mixture a 50 % inhibition was obtained and 10 pM gave a 93 % inhibition. Divalent cations were also strong inhibitors. MgC1, inhibited incorporation by 50 %at 10 mM, MnCl, inhibited the reaction by 80% at 2 mM and 96% at 5 mM, and CaC1, inhibited the methyltransferase by 97 ”/, at 75 mM. Sodium chloride and potassium chloride had no stimulating effect, unlike HeLa cells [lo] and wheat germ [I21 enzymes.

The purified preparation was checked for RNase con- tamination in two different ways. The conversion of uni- formally labeled E. coli RNA to CCl,CO,H-soluble fragments was not observed under standard assay conditions. When bovine lcns polysornes were incubated with the purified enzyme fraction, no endonucleolytic cleavage of the mRNA was detectable by sucrose gradient analysis under conditions where picogram amounts of pancreatic RNase cleaved about 80 % of the polysomes [I 41. The absence of RNase activity is an obvious advantage of this procedure being based upon the use of the VY 1160 yeast strain whose osmotic lysis prevents the liberation of RNase in the supernatant [I 51.

Characterization of Products Methyluted in vitro

Besides commercial GspppsG the enzyme was also tested with undermethylated yeast poly(A)-rich mRNA isolated from an AdoMet auxotrophic mutant strain. Incorporation of radioactivity into CCl,CO,H-insoluble material was depen- dent upon added undermethylated poly(A)-rich mRNA (Fig. 4). The enzyme preparation exhibited a marked pre- ference for undermethylated poly(A)-rich mRNA as opposed

Fig. 3. Time course of cup meihylution. G’ppp”‘G aud [M~-~HlAdoMet were used as acceptor and donor, respectively, and incubation was performed at 30 “C under standard conditions and for the indicated times

. - Z 0 3 -

I

m

W T R N A

Po ldA) - r i ch RNA lpgl

Fig. 4. Enzymatic trunsf2.r qf’ methyl groups OH yeust will-type urirl undermethyluted poly(A)-rich mRNA. The reactions were carried out as described in Materials and Methods. Poly(A)-rich mRNA extracted from strain S288C was used for wild-typc RNA (wT)

to commercial cap analogs ( 5 vs 0.015 methylation) after a 60-mi11 incubation. To characterize further the methylated products, the labeled RNA was extracted with phenol, digested with RNases T, and P, and paper chromatographed as described. Fig. 5 shows the paper chromatography of the methylated cap analogs (Fig. 5A) and the T, and P, digests of the poly(A)-rich mRNA (Fig. 5B). The partially purified enzyme catalyzed the transfer of the methyl group from [Me- ‘HH]AdoMet only to the N-7 position of the cap guanine. When cap analogs were used (Fig. 5A) only one of the two guanines seemed to be methylated since all of the radioactivity was concentrated into one single peak co-migrating with the m7G5’ppp5’G control marker. N o ribose methylation was detectable in either case.

DISCUSSION

The yeast Saccharomyces cerevisiae was previously shown to contain only cap 0 structures [2, 31. We therefore looked for methylase activity in lysed yeast cells in vitro using conditions

Page 5: Partial Purification and Characterization of mRNA (Guanine-7-) Methyltransferase from the Yeast Saccharomyces cerevisiae

121

I A rn7G5'ppp5' m7G5'ppp5'A I

"0 10 20 30 LO Migration (cmi

Fig. 5. Characterization ojnucleotides methylated in vitro. (A) Commercial cap analog Gs'pppsG methylated in vitro and (B) undermethylated yeast poly(A)-rich mRNA were submitted to T, and P, RNase treatments and analysed by paper chromatography on Whatman 3 MM paper as described

similar to those described for other systems (for review see Keith [16]). No activity could be detected in preliminary experiments using different wild-type strains lysed with glass beads even after gel filtration on Sephadex G-25. Only gentle osmotic lysis of the osmotic-sensitive strain VY 1160 provided significant methyl group incorporation into commercial cap analogs. This observation suggests that very strong lysis using glass beads might liberate some factors able to prevent the methylation reaction, as opposed to osmotic lysis which was shown to preserve the structure of some cellular components like lysosomes [15]. Enzyme treatments to generate sphero- plasts amenable to gentle lysis have not been tried. Although commercial cap analogs are specific substrates which can be very conveniently used for enzyme purification, a much higher specific activity (about 300-fold on a molar basis) is observed with undermethylated yeast poly(A)-rich mRNA. This in- dicates that in yeast, as in other organisms [9, 101, structural features of the mRNA chain contribute to the cap-methylation process. No 2-0-ribose methylase activity has been detected,

either in the crude extract no in the purified fraction, which is consistent with the results obtained in viva [2, 31.

Therefore, in contrast to enzyme preparations from animal cells, the yeast system offers the advantage of being completely free from contaminant ribose 2'-O-methyltransferase activity. The complete absence of RNase activity is also an obvious advantage.

Our procedure provides an easy and rapid method for the preparation of mRNA (guanine-7-)methyltransferase. This opens the way to further investigations on yeast mRNA cap methylation mechanisms and its physiological role in transcrip- tion, initiation of translation and RNA stability by a combined biochemical and genetic approach.

We thank H. Bouchet, R. M. Genicot and M. Jadin for the excellent technical assistance and Dr J. Vandenhaute for the helpful discussions. This work was supported by grants from Fonds National de la Recherche Scientifique and Fonds de la Recherche Scientifique Fondamentale Collec- tive. C. Locht holds a pre-doctoral fellowship from the h t i t u f pour SEncouragement de la Recherche Scientifique appliyuee a I'lndustrie et SAgriculture.

REFERENCES

1 . 2.

3.

4. 5.

6.

7. 8. 9.

10. 11.

12.

13. 14. 15. 16.

Banerjee, A. K. (1980) Microbiol. Rev. 44, 175-205. Sripati. L. E., Groner, Y. & Warner, J. R. (1976) J. Biol. Chem. 251,

Mager, W. H., Klootwijk, J. &Klein, I. (1976) Mol. Biol. Reports3,9-

Shatkin, A. J. (1976) Cell, 9, 645-653. Venkov, P. V., Hadjiolov, A. A., Battauer, E. & Schiessinger, D. (1974)

Cherest, H., Surdin-Kerjan, Y., Exinger, F. & Lacroute, F. (1978) Mol.

Bradford, M. M. (1976) A n d . Biochem. 72, 248-254. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685. Martin, S. A. & Moss, B. (1976) J. Biol. Chem. 251, 7313-7321. Ensinger, M. J . & Moss, B. (1976) J . B i d . Chem. 251, 5283-5291. Mizumoto, K. & Lipmann, F. (1979) Proc. Natl Acad. Sci. USA, 76,

Locht, C., Bouchet, H. & Delcour, J. (1982) in Biochemistry of's- Adenosyl-methionine and Related Compounds (Usdin, E., Borchardt, R. T. & Crcveling, C. R., eds) pp. 337-340, MacMillan.

2898-2904.

17.

Biochem. Biophys. Res. Commun. 56, 599 - 604.

Gen. Genet. 163, 153-167.

4961 -4965.

Parks, L. W. & Schlenk, F. (1958) J . Bid. Chem. 230, 295-305. Delcour, J. & Pierssens, A. M. (1979) Ophthalmic Res. 11, 360-365. Venkov, P. V. (1979) Mol. Gen. Genet. 175, 111-112. Kcith, J . M. (1983) CRC Crit. Rev. Biochem. in the press.

C. Locht, J.-L. Beaudart, and J. Delcour, Unite de Genetique Mol6culaire Eucaryotes, Universite Catholique de Louvain, Place de la Croix-du-Sud 5, B-1348 Ottignies/Louvain-la-Neuve, Belgium