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Internal 6-methyladenine residues increase the in vitro translation efficiency of dihydrofolate reductase messenger RNA
Pergamon
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ml. J. Biochem. Cell Bid. Vol. 28, No. 7, pp. 823-829, 1996 Copyright 0 1996 Elsevier Science Ltd S1357-2725@6)000143 Printed in Great Britain. All rights reserved 1357-2725/96 $15.00 + 0.00
Internal 6-Methyladenine Resi s Increase the In Vho Translation Efficiency of Dihydmfolate Reds&se Messenger RNA KEVIN L. HEILMAN,
RICHARD A. LEACH, MARTIN T. TUCK*
Department of Chemistry and the Program in Molecular and Cellular Biology, Ohio University, Athens, OH 45701, U.S.A. N6-Methyladenosine (m6A) is found internaliy in a number of mRNA moiecules from higher eucaryotic cek. In these investigatim, it was found that tbe presenceof m6A resbes increase the in uitro traaflation efficiency of capped T7 transcripts of mouse dihydrofolate redactase (DHFR) mRNA. Using an in vitro rabbit reticulocyte translation system, the formation of internal m6A residueain the DHBR transcripts resubd in a 1.5fold increasein translated DHFR compared to traglcripts void of internal m6A residues. TransIation in a wheat germ system, however, resulted in no increase in translation eflkiency npaa m6A formation, suggest@ that the mechanism may be species-specific.Copyright 0 1996 Elsevier Science Ltd Keywords: 6-Methyladenine
Messenger RNA
Translation
Dihydrofolate reductase
Int. J. Biochem. Cell Biol. (1996) 28, 823-829
event, which occurs at the pre-mRNA before the splicing process. While m6A residues have been observed in the intron regions of premRNA (Aloni et al., 1979; Canaani et al., 1979; Carroll et al., 1990), earlier investigations (Lavi et al., 1977; Chen-Kiang et al., 1979) have demonstrated that most m6A residues in total cellular mRNA are conserved during processing, suggesting that most of the modified residues are located in the exon regions. Probably one of the most puzzling aspects of m6A formation is the nonstoichiometric nature of the reaction. Narayan and Rottman (1988) have shown that in the case of prolactin mRNA only 20% of the transcripts are methylated in viuo and 5% are methylated in an in uitro reaction. While the biological function of m6A formation remains uncertain, a great deal of evidence has suggested that the modification may play a significant role during RNA processing and/or transport. Previous research has demonstrated that treatment of cultured SV40 infected BSC-1 cells (Finkel and Groner, 1983) and B77 chicken embryo fibroblasts (Stoltzfus and Dane, 1982) with the methylation inhibitor cycloleucine caused an accumulation of unspliced pre-RNA
INTRODUCTION
The formation of internal N6-methyladenosine (m6A) residues in certain mRNA molecules is a posttranscriptional modification in which Sadenosyl+methionine (AdoMet) serves as the methyl donor (Tuck, 1992a). Residues of m6A have been found in a number of mRNA molecules from higher eucaryotic organisms (Desrosiers et al., 1974, 1975) as well as plant (Kennedy and Lane, 1979; Haugland and Cline, 1980) and viral (Stoltzfus and Dimock, 1976) systems. While the levels of m6A vary between specific types of transcript, certain messages such as DHFR have been shown to contain high levels of m6A (Rottman et al., 1986) whereas mRNA of globin (Perry and Scherrer, 1975), histone (Moss et al., 1977) and rat albumin (Rottman et al., 1986) are completely void of modified internal adenine residues. The methylation of the internal adenine residues occurs nonrandomly and requires the consensus sequence, Am6AC or Gm6AC (Wei et al., 1976). The formation of m6A is a nuclear *To whom all correspondence Received 5 May 1995; accepted
should be addressed. 8 December 1995. 823
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Kevin
L. Heilman
in the nucleus of both cell types. In addition to the above studies, exposure of HeLa cells with S-tubercidinylhomocysteine, another methylation inhibitor, which is more specific for m6A formation than cycloleucine, was shown to increase the nuclear dwell time of mRNA transcripts by 40% (Camper et al., 1984). In interpreting these experiments, it has been suggested that m6A formation may play a role in the selection of splice sites, and if these selections are disrupted, unprocessed mRNA accumulates in the nucleus and is therefore not properly transported (Aloni et al., 1979; Canaani et al., 1979; Chen-Kiang et al., 1979). Because many mRNA molecules in both cellular and viral systems lack m6A residues, this modification does not appear to be an absolute requirement for the translation of mRNA into protein. The fact that mRNA molecules void of m6A can still be incorporated into intracellular polysomes (Dimock and Stoltzfus, 1979; Kaehler et al., 1979) also supports the above observation. However, no investigations to date have focused on whether the presence of m6A residues alter the translation efficiency (amount of protein product produced) of the mRNA. The present study represents the first investigation of this relationship. Capped T7 transcripts coding for mouse DHFR were methylated in vitro using a partly purified m6A mRNA methyltransferase from HeLa cell nuclei. A comparison was then made between the amount of in vitro translated DHFR from capped methylated transcripts ( + m6A) versus capped unmethylated messages (-m6A). The results demonstrate that the presence of m6A residues were responsible for a 1.5fold increase in the amount of in vitro translated DHFR from a rabbit reticulocyte assay. The amount of DHFR translated was quantitated by trichloroacetic acid (TCA) precipitation and the results were verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Interestingly, the presence of m6A in the transcript had no effect on the amount of in vitro DHFR translated using a wheat germ extract, suggesting that the relationship between m6A formation and translation efficiency may be species-specific. MATERIALS
AND
METHODS
Materials
35S-Methionine (1175 Ci/mmol) and S-[methyl-3H]adenosyl-L-methionine (80 Ci/mmol) were
et al.
purchased from New England Nuclear (Boston, MA). T7 RNA polymerase, in vitro transcription reagents, RNasin, rabbit reticulocyte lysate in vitro translation assay kits and wheat germ extract in vitro translation assay kits were purchased from Promega Biotec (Madison, WI). RNAse-free DNAse was from BoehringerMannheim Biochemicals (Indianapolis, IN). Nuclease Pl, calf intestinal alkaline phosphatase and m7G(5’)ppp(5’)Gm (CAP 1 structure) were purchased from Pharmacia Biotech (Piscataway, NJ). Standard N6-methyladenosine and 2’-O-methylguanosine were obtained from Sigma Chemical Co. (St. Louis, MO). All tissue culture supplies including calf serum were from Life Technologies (Grand Island, NY). Hind III was purchased from New England Biolabs (Beverly, MA). The pDHFR-BS plasmid was kindly provided by Dr Gray F. Crouse, Department of Biology, Emory University, Atlanta, Georgia. pDHFR-BS contains the entire coding region of a mouse DHFR cDNA cloned into the Sma I site of pBluescript (Stratagene). In Vitro
transcription/methylation
of DHFR
mRNA
The following combination assay was performed to prepare internally methylated DHFR mRNA by combining the T7 in vitro transcription assay of DHFR mRNA and the m6A formation assay (Rana and Tuck, 1990). The following reagents were added to achieve a total volume of 100 ~1: 20 ~1 of 5X T7 transcription buffer [200 mM Tris-HCl (pH 7.5), 30mM MgCl,, 10 mM spermidine, and 50 mM NaCl], 10 p1 of 0.1 M dithiothreitol, 10~1 rNTP mix (5 mM CTP, ATP, UTP, and 0.5 mM GTP), 10 ~1 of 5 mM m’(Y)Gppp(5’)Gm (CAP 1 structure), 27 units of RNasin, 10 pg template DNA (pDHFR-BS linearized with Hind III), 40 units of T7 RNA polymerase, 2 ~1 (2.5 PCi) S[methyl-3H]adenosyl-L-methionine, 0.12 mM (final concentration) nonradiolabeled AdoMet, and 25~1 (0.675 pg protein/pi) of partially purified m6A mRNA methyltransferase, the source of which was the major activity peak purified from HeLa cell nuclear extracts through a DEAE cellulose column as described (Tuck, 1992b). The small amount of S-[methyl3H] adenosyl+methionine was added in order to normalize for the amount of m6A formed during the combination assay. The above reaction mixtures were incubated at 37°C for 1 hr. Forty additional units of T7
6-Methyladenine
825
increases translation efficiency
RNA polymerase were then added, and the reaction mixtures incubated for an additional hour at 37°C. The DNA template was digested with RNase-free DNase and the methylated RNA extracted and ethanol precipitated as described (Rana and Tuck, 1990; Tuck, 1992b). The precipitated DHFR transcripts were collected by centrifugation, washed with 70% ethanol, dried in oucuo and dissolved in water treated with diethylpyrocarbonate (DEPC). The concentration of the RNA solutions were routinely determined by measuring absorbance values of diluted samples at 260 nm. The levels of m6A formed were determined by quantitating a small amount (l-2 pg) of the RNA in a liquid scintillation counter. In order to synthesize the control mRNA lacking m6A residues (mock methylated transcripts), the addition of AdoMet (both radiolabeled and nonlabeled) to the assay mixtures was omitted. The remainder of the procedure was as described above. Paper chromatography analysis of the methylated nucleosides from the substrate DHFR mRNA (technique as described below), demonstrated that m6A was the major methylated nucleoside product formed in the combination assay (Fig. 1). This was concluded from the fact that the m6A spot excised from the chromatograph of hydrolyzed DHFR RNA contained 15 times the amount of radioactive methyl functional groups than either the CAP 1 structure or the 2’-O-methylguanosine spots (Fig. 1). It should be noted that radioactivity detected in either of the spots representing the CAP 1 structure (which did not migrate from the origin) or 2’-0-methylguanosine, which would have been expected hydrolysis products if CAP methylation were occurring in the assay, was less than 2-fold background for the scintillation counter. This data therefore demonstrates that the partially purified m6A mRNA methyltransferase used in these investigations is void of significant 7-methylguanine or 2’-0 -methyl mRNA methyltransferase activity and that the increase in translation efficiency observed in these investigations is most likely due to m6A formation. Analysis of the methylated nucleosides formed in the substrate DHFR mRNA The methylated nucleosides formed in the combination transcription/methylation assay described above were analysed using the paper chromatography system described by Harper
et al. (1990). DHFR mRNA was prepared in the combination assay described above, except the amount of [methyl-3H] AdoMet was increased to 10 PCi and the nonradioactive AdoMet was omitted. The ethanol precipitated RNA was dissolved in DEPC-treated water and extracted 5-6 times with a thick slurry of S Sepharose (Sigma) in order to remove any unreacted radiolabeled AdoMet. RNA (20 p g, as determined by absorbance analysis at 260 nm) was digested to nucleotides with 30 pug of nuclease PI in 5 mM sodium acetate (pH 5.2, 45 ,ul total reaction volume) for 5 hr at 37°C. The pH of the reaction mixture was then adjusted to 7.0 by the addition of 1~1 of concentrated NH,OH and 6.6 ,ul of 10 x alkaline phosphatase buffer (supplied with the enzyme). Alkaline phosphatase (22 units) was added to the reaction mixture and the incubation was continued at 37°C for an additional 3 hr. The reaction mixture was then dried in a speed vat concentrator and the residue nucleosides were dissolved in sterile water. Standard N6-methyladenosine, CAP 1 structure and
-.__
1
Rf value
Base
m6A
#
@ 2’.O-mG
m6A 2’.O-mG . cap 1 L
1 ! /
DPM
083
302
0.64
10
--
21
cap 1 structure --+---
2
Fig. 1. Paper chromatography of the methylated nucleosides in DHFR mRNA. DHFR mRNA was transcribed in the presence of partly purified m6A mRNA methyltransferase and [methyl-‘H] AdoMet as described in Materials and Methods. The RNA was purified by ethanol precipitation and hydrolyzed to nucleosides. Nucleosides resulting from the hydrolysis of 8 pg of methylated DHFR RNA (along with unlabeled marker bases) were spotted on a 4 x 16cm sheet of Whatman 3 mm filter paper, which had been treated with 0.4M ammonium sulfate. After chromatography in 76% ethanol, methylated bases were located by UV absorption (of the added unlabeled marker bases) and the spots were excised and radioactivity determined in a liquid scintillation counter. The figure illustrates the position of the marker bases on the chromatograph, R, values of the bases and the amount of radioactivity present in each spot. The reported DPM values are the average of two independent determinations. m6A, N6-methyladenosine; 2’-O-mG, 2’0-methylguanosine; CAP 1 structure, m7GpppGm.
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Kevin L. Heilman et al.
2’-O-methylguanosine were also added to the mixture to serve as chromatography markers. Nucleosides representing 8 ,ug of hydrolyzed RNA were spotted on Whatman 3 mm paper, which had been saturated before use in 0.4 M ammonium sulfate. The chromatographs were developed in 76% ethanol, dried and the standard nucleoside and CAP 1 spots visualized by short-wave UV light. The marker spots were then cut out of the chromatograph and counted in a liquid scintillation counter. Rabbit reticulocyte lysate in vitro translation assay In order to denature the RNA template, the DHFR mRNA was first heated to 65°C for 10 min and then immediately cooled on ice. The rabbit reticulocyte lysate in vitro translation assays were performed using the following reagents/assay: 14 ~1 of rabbit reticulocyte lysate, 0.2 mmol amino acid mixture (minus methionine), 16 units RNasin, 16 y Ci [?S]-methionine, 2.Opg of DHFR mRNA and DEPC-treated H,O in a 20~1 total volume reaction. All reaction tubes were routinely incubated for 1 hr at 30°C. After the incubation, 2-10 ~1 aliquots were removed from each assay tube. The amount of newly synthesized DHFR was then analyzed by TCA precipitation as described below or SDS-PAGE as described in the Promega in vitro translation technical manual (1992) provided with the translation kits. Wheat germ extract in vitro translation assay The DHFR mRNA was heated to 65°C for 10 min and then immediately cooled on ice to denature the template. The wheat germ extract translation reactions were performed using the following reagents/assay: 10~1 of wheat germ extract, 0.8 mmol amino acid mixture (minus methionine), 16 units RNasin, 1OpCi [3”S]methionine, 2.0 pg of DHFR mRNA and DEPC-treated H,O in a 20 ~1 total volume reaction. The reaction tubes were incubated for 1 hr at 25°C. After the incubation, 2-10 ~1 aliquots of the amount of newly synthesized protein were analyzed by TCA precipitation as described below and SDS-PAGE as described elsewhere (Promega Technical Manual, 1992). Trichloroacetic acid (TCA) incorporation assay The amount of newly synthesized DHFR in each translation assay was quantitated by TCA precipitation as described below. Each 2 ~1 aliquot taken from the translation assays was
added to 248 ~1 of 1 N NaOH/2% H,O, in a microcentrifuge tube. For the analysis of wheat germ translation products, the H,O, was omitted from the above solution. The mixtures were then incubated at 37°C for 10 min. After the incubation, 1.0 ml of ice-cold 25% TCA/Z% casamino acids (Difco) was added to each tube. The mixtures were vortexed and allowed to stand on ice for 30 min. The precipitates were collected by filtration through glass fiber filters (Whatman) and washed three times (3 ml/filter) with ice-cold 5% TCA and once with acetone (2 ml/filter). The filters were air dried for 30 min and counted for [35S] incorporation using a liquid scintillation counter.
RESULTS
AND
DISCUSSION
Normalization of the methylated DHFR mRNA for m6A content In this investigation, the effect of m6A content on mRNA translation efficiency was studied using cell-free translation systems. The DHFR transcript used in these investigations contains the entire coding region including the required AUG (start) codon. In order to explore the relationship between m6A content and translation efficiency, it was first necessary to normalize (quantitate) the synthetic DHFR transcripts for m6A levels prior to performing the translation experiments. This was accomplished by adding a small amount (2.5 PCi) of methyl-3H radiolabeled AdoMet to the combination transcription/methylation assay described above. The amount of radioactivity incorporated into l-3 ,ug of DHFR mRNA was then quantitated in a liquid scintillation counter. Based on the specific activity of the AdoMet in the transcription/methylation assay, the amount of m6A formed was calculated. As shown in Fig. 1, at this stage of m6A mRNA methyltransferase purification the major methylated nucleotide formed in the DHFR transcript is m6A. Therefore, the amount of 3H incorporated into the DHFR transcript is mainly due to m6A formation. The results of the normalization experiments demonstrated, on average, that 0.05 pmol of m6A were formed per pmol of RNA. Assuming one m6A residue per transcript, these results would indicate that 5% of the total DHFR transcripts formed in the combination assay contain one m6A residue. these substoichiometric m6A Interestingly, levels are identical to those observed for the
6-Methyladenine
increases translation efficiency
Table 1. Rabbit reticulocyte lysate in viiro translation assay r5S-incorporation Methylated DHFR DPM per assay (pmol) 16537 0.634 Trial 1 23310 0.893 Trial 2 14559 0.558 Trial 3 18135 0.695 Average 3SS-incorporation Mock methylated DHFR DPM per assay (pmol) Trial 1 11588 0.444 Trial 2 13560 0.519 Trial 3 10011 0.384 Average 11719 0.449 Each trial represents the average of TCA precipitable radioactivity from duplicate assay tubes minus control (no added RNA) activity. In all reported trials (from both methylated and mock methylated RNA) different RNA preparations were assayed.
821
incorporation assays were confirmed using SDS-PAGE followed by autoradiography of the gel (Fig. 2). This experiment was critical to confirm that the translation product was in fact full length DHFR protein. As shown in lane 2 of this figure, a band representing DHFR (as confirmed using a DHFR marker; position indicated by the arrow) was synthesized using transcripts containing m6A. This band was also present (but more faint) in the assays in which mock methylated transcripts were translated (lane 3) but was hardly detectable in the control assay (lane 1, no added transcript). The lower molecular weight band present in lanes 2 and 3 is most likely a prematurely terminated translation product of DHFR. Interestingly, the difference in the intensity of the DHFR band comparing methylated and mock methylated transcripts appears to be in vitro methylation of prolactin T7 transcripts greater than the 1.5fold difference observed in (Narayan and Rottman, 1988). the TCA incorporation assay. While the reason for this discrepancy is currently unknown, it Translation of methylated and mock methylated should be noted that although the TCA incorDHFR mRNA using a rabbit reticulocyte in vitro poration assay is a much faster method for translation system translation product analysis, it does suffer from The rabbit reticulocyte lysate translation sys- high background values. This problem is most tem can take advantage of the incorporation of likely due to nonspecific binding of the radio[35S]-methionine to provide qualitative and active label to the precipitated proteins. While quantitative data on protein synthesis. The this background was routinely corrected for in amount of DHFR protein translated in this these studies, it may have effected the results in system was quantitated by measuring the Table 1. In the case of the SDS-PAGE analysis, amount of TCA precipitable [35S]-methionine the fact that this technique does not rely on the incorporated per assay. Initially, the assay con- precipitation of the protein products and separditions were optimized for time and the amount ates molecules based on size and to a certain of RNA (data not shown) using unmethylated, extent electrophoretic mobility, the results from capped DHFR transcripts (DHFR mRNA this method would be immune to the backtranscribed in the absence of AdoMet and ground problem. methyltransferase). The optimum time and Translation of methylated and mock methylated RNA concentration were found to be 60 min DHFR mRNA using a wheat germ in vitro and 2 p g/assay, respectively. As shown in translation system Table 1, methylated DHFR transcripts incorporated an average of 0.695 pmol of methionThe methylated and mock methylated DHFR ine/assay, while mock methylated transcripts mRNA were also translated in wheat germ (transcripts prepared in the absence of AdoMet) extract for comparison. This comparison was incorporated 0.449 pmol/assay. The rabbit retic- made because plant mRNA has been shown to ulocyte translation assay was repeated several contain (on average) a mole percent level of times with different mRNA preparations (each m6A 2-fold less than most animal transcripts reported trial represents a different RNA prep- (Lane and Tumaitis-Kennedy, 1981). The rearation). Analysis of each experimental trial, sults showed that, unlike the rabbit reticulocyte showed that methylated DHFR mRNA always system, there was little difference in the amount translated 1.5 times better than mock methylof DHFR translated using the wheat germ ated DHFR mRNA, even though the amount of extract system (Table 2). As shown in Table 2, “S-methionine incorporation was difficult to proteins translated from methylated DHFR reproduce from trial to trial. mRNA had a specific activity of incorporated The results of the rabbit reticulocyte TCA methionine of 0.265 pmol/assay, while mock
828
Kevin L. Heilman ef
methylated DHFR mRNA had a specific activity of 0.241 pmol/assay. The results of these experiments were also confirmed by SDSPAGE. No difference was seen in the intensity of the DHFR band between methylated and mock methylated transcripts (data not shown). This study has shown that the presence of m6A in DHFR transcripts significantly increases the in vitro translation efficiency of the messages. While the reason for this increase is presently unknown, it can be speculated that m6A residues may stabilize the transcripts in the
al.
assays or contribute to producing a more stable complex with the translating ribosomes. The fact that m6A residues had no effect during wheat germ translation suggests that the mechanism may be unique to mammalian systems. This would not be surprising considering the fact that plant transcripts, on average, contain far less m6A than those of animals (Lane and Tumaitis-Kennedy, 198 1). Finally, in interpreting these results, it should also be noted that synthetic transcripts methylated in vitro (such as those used in these studies) have been shown to have 334-fold less m6A than natural messages (Narayan and Rottman, 1988). This is probably due to the fact that transcripts that have been used during in vitro studies thus far are derived from cDNAs and therefore do not represent unprocessed RNA, the natural substrates for the m6A mRNA methyltransferase. If the fraction of m6A containing DHFR transcripts could be increased to a level that more accurately mimics the in vivo situation (20% in the case of prolactin transcripts; Narayan and Rottman, 1988) the effect of this modification on translation efficiency may have been truly dramatic. In addition, it should also be noted that in order to account for the increase in translation efficiency, considering such a small number of transcripts containing m6A, the individual transcripts containing the modified residues must have an extremely high level of efficiency compared to transcripts void of m6A. It is therefore interesting to speculate if, in fact, the translation efficiency of the DHFR transcripts would have increased even further with an increased level of m6A, and if the in vivo levels of m6A in mRNA, which vary widely Table 2. Wheat germ extract in
Fig. 2. SDS-PAGE/autoradiography of the rabbit reticulocyte lysate translation assay products. Methylated and mock methylated DHFR mRNA (2pg each), which were transcribed in the combination T7-transcription/m6A mRNA methyltransferase assay, were translated using the rabbit reticulocyte lysate in vitro translation system. The newly translated proteins (10~1 of each translation assay) were separated by SDS-PAGE followed by autoradiography. Lane 1: radiolabeled translated products from the rabbit reticulocyte translation assay with no added mRNA. Lane 2: radiolabeled proteins translated from methylated DHFR mRNA. Lane 3: radiolabeled proteins translated from mock methylated DHFR mRNA. The location at which standard bovine liver DHFR (Sigma) migrated during SDS-PAGE (as determined by Coomassie staining of the gel) is marked with an arrow.
translation assay 35S-incorporation per assay (pmol)
vitro
DPM Methylated DHFR Trial 1 8064 0.309 8328 Trial 2 0.319 Trial 3 4340 0.166 Average 6911 0.265 Mock 35S-incorporation methylated DHFR DPM per assay (pmol) Trial 1 6450 0.247 Trial 2 8171 0.313 Trial 3 4251 0.163 Average 6291 0.241 Each trial represents the average of TCA precipitable radioactivity from duplicate assay tubes minus control (no added RNA) activity. In all reported trials (from both methylated and mock methylated RNA) different RNA preparations were assayed.
6-Methyladenine
from transcript to transcript (Tuck, 1992a), would also correlate with translation efficiency. Finally, it should be noted that while this investigation makes claims that m6A levels in mRNA have a positive effect on translation efficiency, owing to the heterogeneous makeup of the methyltransferase preparation used in these studies, we cannot rule out the possibility that other posttranscriptional modifications in the transcripts may be affecting these results. While we have found little evidence for 5’ CAP formation and/or cap modification reactions in the transcripts, which are well known alterations that effect translation efficiency, we cannot rule out the possibility that other modifications to the transcripts that effect translation efficiency are not occurring. REFERENCES
Aloni Y.. Dhar R. and Khoury G. (1979) Methylation of simian virus 40 RNAs. J. Vi&. 32, 5260. Camper S. A., Albers R. J., Coward J. K. and Rottman F. M. (1984) EtTect of undermethylation on mRNA cytoplasmic appearance and half-life. Mol. Cell. Biol. 4, 538-543. Canaani D., Kahana C., Lavi S. and Groner Y. (1979) Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA. Nucleic Acids Rex
6, 2879-2899.
Carroll S. M., Narayan P. and Rottman F. M. (1990) N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA. Mol. Cell Biol. 10, 44564465. Chen-Kiang S., Nevins J. R. and Darnell J. E. Jr (1979) 6-Methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA. J. Mol. Biol.
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Desrosiers R. C.. Friderici K. H. and Rottman F. (1974) Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc. Nat1 Acad. Sci. 71, 3971-3975.
Desrosiers R. C., Friderici K. H. and Rottman F. M. (1975) Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5’ terminus. Biochemistry
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14, 43674374.
Dimock K. and Stoltzfus C. M. (1979) Processing and function of undermethylated chicken embryo fibroblast mRNA. J. Biol. Chem. 254, 5591-5594. Finkel D. and Groner Y. (1983) Methylation of adenosine residues (m6A) in pre-mRNA are important for formation of late simian virus 40 mRNAs. Virology 131, 409425. Harper J. E., Miceli S. M., Roberts R. J. and Manley J. L. (1990) Sequence specificity of the human mRNA N6-
adenosine methylase in t&o. Nucleic Acid.5 Res. 18, 573555741. Haugland R. A. and Cline M. G. (1980) Post-transcriptional modifications of oat coleoptile ribonucleic acids. 5’terminal capping and methylation of internal nucleosides in poly(A)-rich RNA. Eur. J. Biochem, 104, 271-277. Kaehler M., Coward J. and Rottman F. (1979) Cytoplasmic location of undermethylated messenger RNA in Novikoff cells. Nucleic Acid? Res. 6, 1161-1175. Kennedy T. D. and Lane B. G. (1979) Wheat embryo ribonucleates XIII. Methyl-substituted nucleoside constituents and 5’ terminal dinucleotide sequences in bulk poly(A) rich RNA from imbibing wheat embryos. Chn. J. Biochem.
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Lane B. G. and Tumaitis-Kennedy T. D. (1981) Comparative study of levels of secondary processing in bulk mRNA from dry and germinating wheat embryos. Eur. J. Biochem.
114, 457-463.
Lavi Il., Fernandez-Munoz R. and Darnell Jr. J. E. (1977) Content of N-6 methyladenylic acid in heterogeneous nuclear and messenger RNA in HeLa cells. Nuclek Acids Res. 4, 63-69.
Moss B., Gershowitz A., Weber L. A. and Baglioni C. (1977) Histone mRNAs contain blocked and methylated 5’ terminal sequences but lack methylated nucleosides at internal positions. Cell 10, 113-120. Narayan P. and Rottman F. M. (1988) An in uitro system for accurate methylation of internal adenine residues in messenger RNA. Science 242, 1159-I 162. Perry R. P. and Scherrer K. (1975) The methylated constituents of globin mRNA. FEBS Lett. 57, 73.-78. Promega Technical Manual, Translation In Vitro. 1992. Rana A. P. and Tuck M. T. (1990) Analysis and in uitro localization of internal methylated adenine residues in dihydrofolate reductase mRNA. Nucleic Acids Res. 18, 4803-4807. Rottman F., Naraydn P., Goodwin R., Camper S., Yao Y., Horowitz S., Albers R., Ayers D., Maroney P. and Nilson T. (1986) Distribution of m6A in RNA and its possible biological role. In Biological Methylation and Drug Design (Edited by R. T. Borchardt, C. R. Creveling and P. M. Ueland), pp. 1899200. Humana Press, Clifton, NJ. Stoltzfus C. M. and Dane R. W. (1982) Accumulation of spliced avian retrovirus mRNA is inhibited in S-adenosyl-methionine-depleted chicken embryo fibroblasts. J. Viral. 42, 9188931. Stoltzfus C. M. and Dimock K. (1976) Evidence for methylation of B77 avian sarcoma virus genome RNA subunits. J. Viral. 18, 586-595. Tuck M. T. (1992a) Minireview-The formation of internal 6-methyladenine residues in eucaryotic messenger RNA. Int.
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Tuck M. T. (1992b) Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues. Biochem. J. 288, 233-240. Wei C. M., Gershowitz A. and Moss B. (1976) 5’-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA. Biochemistry 15, 397-401.
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ml. J. Biochem. Cell Bid. Vol. 28, No. 7, pp. 823-829, 1996 Copyright 0 1996 Elsevier Science Ltd S1357-2725@6)000143 Printed in Great Britain. All rights reserved 1357-2725/96 $15.00 + 0.00
Internal 6-Methyladenine Resi s Increase the In Vho Translation Efficiency of Dihydmfolate Reds&se Messenger RNA KEVIN L. HEILMAN,
RICHARD A. LEACH, MARTIN T. TUCK*
Department of Chemistry and the Program in Molecular and Cellular Biology, Ohio University, Athens, OH 45701, U.S.A. N6-Methyladenosine (m6A) is found internaliy in a number of mRNA moiecules from higher eucaryotic cek. In these investigatim, it was found that tbe presenceof m6A resbes increase the in uitro traaflation efficiency of capped T7 transcripts of mouse dihydrofolate redactase (DHFR) mRNA. Using an in vitro rabbit reticulocyte translation system, the formation of internal m6A residueain the DHBR transcripts resubd in a 1.5fold increasein translated DHFR compared to traglcripts void of internal m6A residues. TransIation in a wheat germ system, however, resulted in no increase in translation eflkiency npaa m6A formation, suggest@ that the mechanism may be species-specific.Copyright 0 1996 Elsevier Science Ltd Keywords: 6-Methyladenine
Messenger RNA
Translation
Dihydrofolate reductase
Int. J. Biochem. Cell Biol. (1996) 28, 823-829
event, which occurs at the pre-mRNA before the splicing process. While m6A residues have been observed in the intron regions of premRNA (Aloni et al., 1979; Canaani et al., 1979; Carroll et al., 1990), earlier investigations (Lavi et al., 1977; Chen-Kiang et al., 1979) have demonstrated that most m6A residues in total cellular mRNA are conserved during processing, suggesting that most of the modified residues are located in the exon regions. Probably one of the most puzzling aspects of m6A formation is the nonstoichiometric nature of the reaction. Narayan and Rottman (1988) have shown that in the case of prolactin mRNA only 20% of the transcripts are methylated in viuo and 5% are methylated in an in uitro reaction. While the biological function of m6A formation remains uncertain, a great deal of evidence has suggested that the modification may play a significant role during RNA processing and/or transport. Previous research has demonstrated that treatment of cultured SV40 infected BSC-1 cells (Finkel and Groner, 1983) and B77 chicken embryo fibroblasts (Stoltzfus and Dane, 1982) with the methylation inhibitor cycloleucine caused an accumulation of unspliced pre-RNA
INTRODUCTION
The formation of internal N6-methyladenosine (m6A) residues in certain mRNA molecules is a posttranscriptional modification in which Sadenosyl+methionine (AdoMet) serves as the methyl donor (Tuck, 1992a). Residues of m6A have been found in a number of mRNA molecules from higher eucaryotic organisms (Desrosiers et al., 1974, 1975) as well as plant (Kennedy and Lane, 1979; Haugland and Cline, 1980) and viral (Stoltzfus and Dimock, 1976) systems. While the levels of m6A vary between specific types of transcript, certain messages such as DHFR have been shown to contain high levels of m6A (Rottman et al., 1986) whereas mRNA of globin (Perry and Scherrer, 1975), histone (Moss et al., 1977) and rat albumin (Rottman et al., 1986) are completely void of modified internal adenine residues. The methylation of the internal adenine residues occurs nonrandomly and requires the consensus sequence, Am6AC or Gm6AC (Wei et al., 1976). The formation of m6A is a nuclear *To whom all correspondence Received 5 May 1995; accepted
should be addressed. 8 December 1995. 823
824
Kevin
L. Heilman
in the nucleus of both cell types. In addition to the above studies, exposure of HeLa cells with S-tubercidinylhomocysteine, another methylation inhibitor, which is more specific for m6A formation than cycloleucine, was shown to increase the nuclear dwell time of mRNA transcripts by 40% (Camper et al., 1984). In interpreting these experiments, it has been suggested that m6A formation may play a role in the selection of splice sites, and if these selections are disrupted, unprocessed mRNA accumulates in the nucleus and is therefore not properly transported (Aloni et al., 1979; Canaani et al., 1979; Chen-Kiang et al., 1979). Because many mRNA molecules in both cellular and viral systems lack m6A residues, this modification does not appear to be an absolute requirement for the translation of mRNA into protein. The fact that mRNA molecules void of m6A can still be incorporated into intracellular polysomes (Dimock and Stoltzfus, 1979; Kaehler et al., 1979) also supports the above observation. However, no investigations to date have focused on whether the presence of m6A residues alter the translation efficiency (amount of protein product produced) of the mRNA. The present study represents the first investigation of this relationship. Capped T7 transcripts coding for mouse DHFR were methylated in vitro using a partly purified m6A mRNA methyltransferase from HeLa cell nuclei. A comparison was then made between the amount of in vitro translated DHFR from capped methylated transcripts ( + m6A) versus capped unmethylated messages (-m6A). The results demonstrate that the presence of m6A residues were responsible for a 1.5fold increase in the amount of in vitro translated DHFR from a rabbit reticulocyte assay. The amount of DHFR translated was quantitated by trichloroacetic acid (TCA) precipitation and the results were verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Interestingly, the presence of m6A in the transcript had no effect on the amount of in vitro DHFR translated using a wheat germ extract, suggesting that the relationship between m6A formation and translation efficiency may be species-specific. MATERIALS
AND
METHODS
Materials
35S-Methionine (1175 Ci/mmol) and S-[methyl-3H]adenosyl-L-methionine (80 Ci/mmol) were
et al.
purchased from New England Nuclear (Boston, MA). T7 RNA polymerase, in vitro transcription reagents, RNasin, rabbit reticulocyte lysate in vitro translation assay kits and wheat germ extract in vitro translation assay kits were purchased from Promega Biotec (Madison, WI). RNAse-free DNAse was from BoehringerMannheim Biochemicals (Indianapolis, IN). Nuclease Pl, calf intestinal alkaline phosphatase and m7G(5’)ppp(5’)Gm (CAP 1 structure) were purchased from Pharmacia Biotech (Piscataway, NJ). Standard N6-methyladenosine and 2’-O-methylguanosine were obtained from Sigma Chemical Co. (St. Louis, MO). All tissue culture supplies including calf serum were from Life Technologies (Grand Island, NY). Hind III was purchased from New England Biolabs (Beverly, MA). The pDHFR-BS plasmid was kindly provided by Dr Gray F. Crouse, Department of Biology, Emory University, Atlanta, Georgia. pDHFR-BS contains the entire coding region of a mouse DHFR cDNA cloned into the Sma I site of pBluescript (Stratagene). In Vitro
transcription/methylation
of DHFR
mRNA
The following combination assay was performed to prepare internally methylated DHFR mRNA by combining the T7 in vitro transcription assay of DHFR mRNA and the m6A formation assay (Rana and Tuck, 1990). The following reagents were added to achieve a total volume of 100 ~1: 20 ~1 of 5X T7 transcription buffer [200 mM Tris-HCl (pH 7.5), 30mM MgCl,, 10 mM spermidine, and 50 mM NaCl], 10 p1 of 0.1 M dithiothreitol, 10~1 rNTP mix (5 mM CTP, ATP, UTP, and 0.5 mM GTP), 10 ~1 of 5 mM m’(Y)Gppp(5’)Gm (CAP 1 structure), 27 units of RNasin, 10 pg template DNA (pDHFR-BS linearized with Hind III), 40 units of T7 RNA polymerase, 2 ~1 (2.5 PCi) S[methyl-3H]adenosyl-L-methionine, 0.12 mM (final concentration) nonradiolabeled AdoMet, and 25~1 (0.675 pg protein/pi) of partially purified m6A mRNA methyltransferase, the source of which was the major activity peak purified from HeLa cell nuclear extracts through a DEAE cellulose column as described (Tuck, 1992b). The small amount of S-[methyl3H] adenosyl+methionine was added in order to normalize for the amount of m6A formed during the combination assay. The above reaction mixtures were incubated at 37°C for 1 hr. Forty additional units of T7
6-Methyladenine
825
increases translation efficiency
RNA polymerase were then added, and the reaction mixtures incubated for an additional hour at 37°C. The DNA template was digested with RNase-free DNase and the methylated RNA extracted and ethanol precipitated as described (Rana and Tuck, 1990; Tuck, 1992b). The precipitated DHFR transcripts were collected by centrifugation, washed with 70% ethanol, dried in oucuo and dissolved in water treated with diethylpyrocarbonate (DEPC). The concentration of the RNA solutions were routinely determined by measuring absorbance values of diluted samples at 260 nm. The levels of m6A formed were determined by quantitating a small amount (l-2 pg) of the RNA in a liquid scintillation counter. In order to synthesize the control mRNA lacking m6A residues (mock methylated transcripts), the addition of AdoMet (both radiolabeled and nonlabeled) to the assay mixtures was omitted. The remainder of the procedure was as described above. Paper chromatography analysis of the methylated nucleosides from the substrate DHFR mRNA (technique as described below), demonstrated that m6A was the major methylated nucleoside product formed in the combination assay (Fig. 1). This was concluded from the fact that the m6A spot excised from the chromatograph of hydrolyzed DHFR RNA contained 15 times the amount of radioactive methyl functional groups than either the CAP 1 structure or the 2’-O-methylguanosine spots (Fig. 1). It should be noted that radioactivity detected in either of the spots representing the CAP 1 structure (which did not migrate from the origin) or 2’-0-methylguanosine, which would have been expected hydrolysis products if CAP methylation were occurring in the assay, was less than 2-fold background for the scintillation counter. This data therefore demonstrates that the partially purified m6A mRNA methyltransferase used in these investigations is void of significant 7-methylguanine or 2’-0 -methyl mRNA methyltransferase activity and that the increase in translation efficiency observed in these investigations is most likely due to m6A formation. Analysis of the methylated nucleosides formed in the substrate DHFR mRNA The methylated nucleosides formed in the combination transcription/methylation assay described above were analysed using the paper chromatography system described by Harper
et al. (1990). DHFR mRNA was prepared in the combination assay described above, except the amount of [methyl-3H] AdoMet was increased to 10 PCi and the nonradioactive AdoMet was omitted. The ethanol precipitated RNA was dissolved in DEPC-treated water and extracted 5-6 times with a thick slurry of S Sepharose (Sigma) in order to remove any unreacted radiolabeled AdoMet. RNA (20 p g, as determined by absorbance analysis at 260 nm) was digested to nucleotides with 30 pug of nuclease PI in 5 mM sodium acetate (pH 5.2, 45 ,ul total reaction volume) for 5 hr at 37°C. The pH of the reaction mixture was then adjusted to 7.0 by the addition of 1~1 of concentrated NH,OH and 6.6 ,ul of 10 x alkaline phosphatase buffer (supplied with the enzyme). Alkaline phosphatase (22 units) was added to the reaction mixture and the incubation was continued at 37°C for an additional 3 hr. The reaction mixture was then dried in a speed vat concentrator and the residue nucleosides were dissolved in sterile water. Standard N6-methyladenosine, CAP 1 structure and
-.__
1
Rf value
Base
m6A
#
@ 2’.O-mG
m6A 2’.O-mG . cap 1 L
1 ! /
DPM
083
302
0.64
10
--
21
cap 1 structure --+---
2
Fig. 1. Paper chromatography of the methylated nucleosides in DHFR mRNA. DHFR mRNA was transcribed in the presence of partly purified m6A mRNA methyltransferase and [methyl-‘H] AdoMet as described in Materials and Methods. The RNA was purified by ethanol precipitation and hydrolyzed to nucleosides. Nucleosides resulting from the hydrolysis of 8 pg of methylated DHFR RNA (along with unlabeled marker bases) were spotted on a 4 x 16cm sheet of Whatman 3 mm filter paper, which had been treated with 0.4M ammonium sulfate. After chromatography in 76% ethanol, methylated bases were located by UV absorption (of the added unlabeled marker bases) and the spots were excised and radioactivity determined in a liquid scintillation counter. The figure illustrates the position of the marker bases on the chromatograph, R, values of the bases and the amount of radioactivity present in each spot. The reported DPM values are the average of two independent determinations. m6A, N6-methyladenosine; 2’-O-mG, 2’0-methylguanosine; CAP 1 structure, m7GpppGm.
826
Kevin L. Heilman et al.
2’-O-methylguanosine were also added to the mixture to serve as chromatography markers. Nucleosides representing 8 ,ug of hydrolyzed RNA were spotted on Whatman 3 mm paper, which had been saturated before use in 0.4 M ammonium sulfate. The chromatographs were developed in 76% ethanol, dried and the standard nucleoside and CAP 1 spots visualized by short-wave UV light. The marker spots were then cut out of the chromatograph and counted in a liquid scintillation counter. Rabbit reticulocyte lysate in vitro translation assay In order to denature the RNA template, the DHFR mRNA was first heated to 65°C for 10 min and then immediately cooled on ice. The rabbit reticulocyte lysate in vitro translation assays were performed using the following reagents/assay: 14 ~1 of rabbit reticulocyte lysate, 0.2 mmol amino acid mixture (minus methionine), 16 units RNasin, 16 y Ci [?S]-methionine, 2.Opg of DHFR mRNA and DEPC-treated H,O in a 20~1 total volume reaction. All reaction tubes were routinely incubated for 1 hr at 30°C. After the incubation, 2-10 ~1 aliquots were removed from each assay tube. The amount of newly synthesized DHFR was then analyzed by TCA precipitation as described below or SDS-PAGE as described in the Promega in vitro translation technical manual (1992) provided with the translation kits. Wheat germ extract in vitro translation assay The DHFR mRNA was heated to 65°C for 10 min and then immediately cooled on ice to denature the template. The wheat germ extract translation reactions were performed using the following reagents/assay: 10~1 of wheat germ extract, 0.8 mmol amino acid mixture (minus methionine), 16 units RNasin, 1OpCi [3”S]methionine, 2.0 pg of DHFR mRNA and DEPC-treated H,O in a 20 ~1 total volume reaction. The reaction tubes were incubated for 1 hr at 25°C. After the incubation, 2-10 ~1 aliquots of the amount of newly synthesized protein were analyzed by TCA precipitation as described below and SDS-PAGE as described elsewhere (Promega Technical Manual, 1992). Trichloroacetic acid (TCA) incorporation assay The amount of newly synthesized DHFR in each translation assay was quantitated by TCA precipitation as described below. Each 2 ~1 aliquot taken from the translation assays was
added to 248 ~1 of 1 N NaOH/2% H,O, in a microcentrifuge tube. For the analysis of wheat germ translation products, the H,O, was omitted from the above solution. The mixtures were then incubated at 37°C for 10 min. After the incubation, 1.0 ml of ice-cold 25% TCA/Z% casamino acids (Difco) was added to each tube. The mixtures were vortexed and allowed to stand on ice for 30 min. The precipitates were collected by filtration through glass fiber filters (Whatman) and washed three times (3 ml/filter) with ice-cold 5% TCA and once with acetone (2 ml/filter). The filters were air dried for 30 min and counted for [35S] incorporation using a liquid scintillation counter.
RESULTS
AND
DISCUSSION
Normalization of the methylated DHFR mRNA for m6A content In this investigation, the effect of m6A content on mRNA translation efficiency was studied using cell-free translation systems. The DHFR transcript used in these investigations contains the entire coding region including the required AUG (start) codon. In order to explore the relationship between m6A content and translation efficiency, it was first necessary to normalize (quantitate) the synthetic DHFR transcripts for m6A levels prior to performing the translation experiments. This was accomplished by adding a small amount (2.5 PCi) of methyl-3H radiolabeled AdoMet to the combination transcription/methylation assay described above. The amount of radioactivity incorporated into l-3 ,ug of DHFR mRNA was then quantitated in a liquid scintillation counter. Based on the specific activity of the AdoMet in the transcription/methylation assay, the amount of m6A formed was calculated. As shown in Fig. 1, at this stage of m6A mRNA methyltransferase purification the major methylated nucleotide formed in the DHFR transcript is m6A. Therefore, the amount of 3H incorporated into the DHFR transcript is mainly due to m6A formation. The results of the normalization experiments demonstrated, on average, that 0.05 pmol of m6A were formed per pmol of RNA. Assuming one m6A residue per transcript, these results would indicate that 5% of the total DHFR transcripts formed in the combination assay contain one m6A residue. these substoichiometric m6A Interestingly, levels are identical to those observed for the
6-Methyladenine
increases translation efficiency
Table 1. Rabbit reticulocyte lysate in viiro translation assay r5S-incorporation Methylated DHFR DPM per assay (pmol) 16537 0.634 Trial 1 23310 0.893 Trial 2 14559 0.558 Trial 3 18135 0.695 Average 3SS-incorporation Mock methylated DHFR DPM per assay (pmol) Trial 1 11588 0.444 Trial 2 13560 0.519 Trial 3 10011 0.384 Average 11719 0.449 Each trial represents the average of TCA precipitable radioactivity from duplicate assay tubes minus control (no added RNA) activity. In all reported trials (from both methylated and mock methylated RNA) different RNA preparations were assayed.
821
incorporation assays were confirmed using SDS-PAGE followed by autoradiography of the gel (Fig. 2). This experiment was critical to confirm that the translation product was in fact full length DHFR protein. As shown in lane 2 of this figure, a band representing DHFR (as confirmed using a DHFR marker; position indicated by the arrow) was synthesized using transcripts containing m6A. This band was also present (but more faint) in the assays in which mock methylated transcripts were translated (lane 3) but was hardly detectable in the control assay (lane 1, no added transcript). The lower molecular weight band present in lanes 2 and 3 is most likely a prematurely terminated translation product of DHFR. Interestingly, the difference in the intensity of the DHFR band comparing methylated and mock methylated transcripts appears to be in vitro methylation of prolactin T7 transcripts greater than the 1.5fold difference observed in (Narayan and Rottman, 1988). the TCA incorporation assay. While the reason for this discrepancy is currently unknown, it Translation of methylated and mock methylated should be noted that although the TCA incorDHFR mRNA using a rabbit reticulocyte in vitro poration assay is a much faster method for translation system translation product analysis, it does suffer from The rabbit reticulocyte lysate translation sys- high background values. This problem is most tem can take advantage of the incorporation of likely due to nonspecific binding of the radio[35S]-methionine to provide qualitative and active label to the precipitated proteins. While quantitative data on protein synthesis. The this background was routinely corrected for in amount of DHFR protein translated in this these studies, it may have effected the results in system was quantitated by measuring the Table 1. In the case of the SDS-PAGE analysis, amount of TCA precipitable [35S]-methionine the fact that this technique does not rely on the incorporated per assay. Initially, the assay con- precipitation of the protein products and separditions were optimized for time and the amount ates molecules based on size and to a certain of RNA (data not shown) using unmethylated, extent electrophoretic mobility, the results from capped DHFR transcripts (DHFR mRNA this method would be immune to the backtranscribed in the absence of AdoMet and ground problem. methyltransferase). The optimum time and Translation of methylated and mock methylated RNA concentration were found to be 60 min DHFR mRNA using a wheat germ in vitro and 2 p g/assay, respectively. As shown in translation system Table 1, methylated DHFR transcripts incorporated an average of 0.695 pmol of methionThe methylated and mock methylated DHFR ine/assay, while mock methylated transcripts mRNA were also translated in wheat germ (transcripts prepared in the absence of AdoMet) extract for comparison. This comparison was incorporated 0.449 pmol/assay. The rabbit retic- made because plant mRNA has been shown to ulocyte translation assay was repeated several contain (on average) a mole percent level of times with different mRNA preparations (each m6A 2-fold less than most animal transcripts reported trial represents a different RNA prep- (Lane and Tumaitis-Kennedy, 1981). The rearation). Analysis of each experimental trial, sults showed that, unlike the rabbit reticulocyte showed that methylated DHFR mRNA always system, there was little difference in the amount translated 1.5 times better than mock methylof DHFR translated using the wheat germ ated DHFR mRNA, even though the amount of extract system (Table 2). As shown in Table 2, “S-methionine incorporation was difficult to proteins translated from methylated DHFR reproduce from trial to trial. mRNA had a specific activity of incorporated The results of the rabbit reticulocyte TCA methionine of 0.265 pmol/assay, while mock
828
Kevin L. Heilman ef
methylated DHFR mRNA had a specific activity of 0.241 pmol/assay. The results of these experiments were also confirmed by SDSPAGE. No difference was seen in the intensity of the DHFR band between methylated and mock methylated transcripts (data not shown). This study has shown that the presence of m6A in DHFR transcripts significantly increases the in vitro translation efficiency of the messages. While the reason for this increase is presently unknown, it can be speculated that m6A residues may stabilize the transcripts in the
al.
assays or contribute to producing a more stable complex with the translating ribosomes. The fact that m6A residues had no effect during wheat germ translation suggests that the mechanism may be unique to mammalian systems. This would not be surprising considering the fact that plant transcripts, on average, contain far less m6A than those of animals (Lane and Tumaitis-Kennedy, 198 1). Finally, in interpreting these results, it should also be noted that synthetic transcripts methylated in vitro (such as those used in these studies) have been shown to have 334-fold less m6A than natural messages (Narayan and Rottman, 1988). This is probably due to the fact that transcripts that have been used during in vitro studies thus far are derived from cDNAs and therefore do not represent unprocessed RNA, the natural substrates for the m6A mRNA methyltransferase. If the fraction of m6A containing DHFR transcripts could be increased to a level that more accurately mimics the in vivo situation (20% in the case of prolactin transcripts; Narayan and Rottman, 1988) the effect of this modification on translation efficiency may have been truly dramatic. In addition, it should also be noted that in order to account for the increase in translation efficiency, considering such a small number of transcripts containing m6A, the individual transcripts containing the modified residues must have an extremely high level of efficiency compared to transcripts void of m6A. It is therefore interesting to speculate if, in fact, the translation efficiency of the DHFR transcripts would have increased even further with an increased level of m6A, and if the in vivo levels of m6A in mRNA, which vary widely Table 2. Wheat germ extract in
Fig. 2. SDS-PAGE/autoradiography of the rabbit reticulocyte lysate translation assay products. Methylated and mock methylated DHFR mRNA (2pg each), which were transcribed in the combination T7-transcription/m6A mRNA methyltransferase assay, were translated using the rabbit reticulocyte lysate in vitro translation system. The newly translated proteins (10~1 of each translation assay) were separated by SDS-PAGE followed by autoradiography. Lane 1: radiolabeled translated products from the rabbit reticulocyte translation assay with no added mRNA. Lane 2: radiolabeled proteins translated from methylated DHFR mRNA. Lane 3: radiolabeled proteins translated from mock methylated DHFR mRNA. The location at which standard bovine liver DHFR (Sigma) migrated during SDS-PAGE (as determined by Coomassie staining of the gel) is marked with an arrow.
translation assay 35S-incorporation per assay (pmol)
vitro
DPM Methylated DHFR Trial 1 8064 0.309 8328 Trial 2 0.319 Trial 3 4340 0.166 Average 6911 0.265 Mock 35S-incorporation methylated DHFR DPM per assay (pmol) Trial 1 6450 0.247 Trial 2 8171 0.313 Trial 3 4251 0.163 Average 6291 0.241 Each trial represents the average of TCA precipitable radioactivity from duplicate assay tubes minus control (no added RNA) activity. In all reported trials (from both methylated and mock methylated RNA) different RNA preparations were assayed.
6-Methyladenine
from transcript to transcript (Tuck, 1992a), would also correlate with translation efficiency. Finally, it should be noted that while this investigation makes claims that m6A levels in mRNA have a positive effect on translation efficiency, owing to the heterogeneous makeup of the methyltransferase preparation used in these studies, we cannot rule out the possibility that other posttranscriptional modifications in the transcripts may be affecting these results. While we have found little evidence for 5’ CAP formation and/or cap modification reactions in the transcripts, which are well known alterations that effect translation efficiency, we cannot rule out the possibility that other modifications to the transcripts that effect translation efficiency are not occurring. REFERENCES
Aloni Y.. Dhar R. and Khoury G. (1979) Methylation of simian virus 40 RNAs. J. Vi&. 32, 5260. Camper S. A., Albers R. J., Coward J. K. and Rottman F. M. (1984) EtTect of undermethylation on mRNA cytoplasmic appearance and half-life. Mol. Cell. Biol. 4, 538-543. Canaani D., Kahana C., Lavi S. and Groner Y. (1979) Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA. Nucleic Acids Rex
6, 2879-2899.
Carroll S. M., Narayan P. and Rottman F. M. (1990) N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA. Mol. Cell Biol. 10, 44564465. Chen-Kiang S., Nevins J. R. and Darnell J. E. Jr (1979) 6-Methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA. J. Mol. Biol.
135, 733-752.
Desrosiers R. C.. Friderici K. H. and Rottman F. (1974) Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc. Nat1 Acad. Sci. 71, 3971-3975.
Desrosiers R. C., Friderici K. H. and Rottman F. M. (1975) Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5’ terminus. Biochemistry
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increases translation efficiency
14, 43674374.
Dimock K. and Stoltzfus C. M. (1979) Processing and function of undermethylated chicken embryo fibroblast mRNA. J. Biol. Chem. 254, 5591-5594. Finkel D. and Groner Y. (1983) Methylation of adenosine residues (m6A) in pre-mRNA are important for formation of late simian virus 40 mRNAs. Virology 131, 409425. Harper J. E., Miceli S. M., Roberts R. J. and Manley J. L. (1990) Sequence specificity of the human mRNA N6-
adenosine methylase in t&o. Nucleic Acid.5 Res. 18, 573555741. Haugland R. A. and Cline M. G. (1980) Post-transcriptional modifications of oat coleoptile ribonucleic acids. 5’terminal capping and methylation of internal nucleosides in poly(A)-rich RNA. Eur. J. Biochem, 104, 271-277. Kaehler M., Coward J. and Rottman F. (1979) Cytoplasmic location of undermethylated messenger RNA in Novikoff cells. Nucleic Acid? Res. 6, 1161-1175. Kennedy T. D. and Lane B. G. (1979) Wheat embryo ribonucleates XIII. Methyl-substituted nucleoside constituents and 5’ terminal dinucleotide sequences in bulk poly(A) rich RNA from imbibing wheat embryos. Chn. J. Biochem.
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Lane B. G. and Tumaitis-Kennedy T. D. (1981) Comparative study of levels of secondary processing in bulk mRNA from dry and germinating wheat embryos. Eur. J. Biochem.
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Lavi Il., Fernandez-Munoz R. and Darnell Jr. J. E. (1977) Content of N-6 methyladenylic acid in heterogeneous nuclear and messenger RNA in HeLa cells. Nuclek Acids Res. 4, 63-69.
Moss B., Gershowitz A., Weber L. A. and Baglioni C. (1977) Histone mRNAs contain blocked and methylated 5’ terminal sequences but lack methylated nucleosides at internal positions. Cell 10, 113-120. Narayan P. and Rottman F. M. (1988) An in uitro system for accurate methylation of internal adenine residues in messenger RNA. Science 242, 1159-I 162. Perry R. P. and Scherrer K. (1975) The methylated constituents of globin mRNA. FEBS Lett. 57, 73.-78. Promega Technical Manual, Translation In Vitro. 1992. Rana A. P. and Tuck M. T. (1990) Analysis and in uitro localization of internal methylated adenine residues in dihydrofolate reductase mRNA. Nucleic Acids Res. 18, 4803-4807. Rottman F., Naraydn P., Goodwin R., Camper S., Yao Y., Horowitz S., Albers R., Ayers D., Maroney P. and Nilson T. (1986) Distribution of m6A in RNA and its possible biological role. In Biological Methylation and Drug Design (Edited by R. T. Borchardt, C. R. Creveling and P. M. Ueland), pp. 1899200. Humana Press, Clifton, NJ. Stoltzfus C. M. and Dane R. W. (1982) Accumulation of spliced avian retrovirus mRNA is inhibited in S-adenosyl-methionine-depleted chicken embryo fibroblasts. J. Viral. 42, 9188931. Stoltzfus C. M. and Dimock K. (1976) Evidence for methylation of B77 avian sarcoma virus genome RNA subunits. J. Viral. 18, 586-595. Tuck M. T. (1992a) Minireview-The formation of internal 6-methyladenine residues in eucaryotic messenger RNA. Int.
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Tuck M. T. (1992b) Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues. Biochem. J. 288, 233-240. Wei C. M., Gershowitz A. and Moss B. (1976) 5’-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA. Biochemistry 15, 397-401.