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mRNA Methylation and Protein Synthesis in Extracts from Embryos of Brine Shrimp, Artemia salinu

Cell-free protein-synthesizing extracts prepared from the brine shrimp, Artemia salina, translate methylated mRNAs. Reovirus unmethylated mRNA is inactive as a template when methylation is prevented by the inhibitor, S-adenosylhomocysteine. A salina mRNAs from both undeveloped and developed embryos contain 5'-terminal 7-methylguanosine in an inverted 5'-5' linkage through three phosphate groups to the rest of the polynucleotide chain. Removal of the 7-methylguanosine by beta elimination converts the mRNA from an active form to one inactive in protein synthesis in extracts of A. salina or wheat germ. Extracts of undeveloped and developed embryos methylate reovirus unmethylated mRNA at the 5' ends to form 5'-terminal structures of the type, m7G(5')ppp(5')G and m7G(5')ppp(5')Gm.

Removal of the 7-methylguanosine by fi elimination converts the mRNA from an active form to one inactive in protein synthesis in extracts of A. salina or wheat germ.
Unfertilized eggs of sea urchin (l-3) and Xenopus laeuis (4,5) contain latent "maternal" mRNA that is synthesized during oogenesis. Despite the presence of mRNA, there is little protein synthesis in mature unfertilized eggs or in egg homogenates (1). Upon fertilization and during the early stages of development, the stored mRNA becomes an active template for protein synthesis. A similar activation of protein synthesis has been observed in dry wheat embryos (6,7). Ungerminated soybean seeds (8) and encysted embryos of the brine shrimp, Artemia salina (g-11), were also found to contain stored mRNA. Several mechanisms for regulating maternal mRNA expression during development have been proposed. For example, it has been suggested that latent mRNA may be complexed with proteins that can be removed after fertilization (1,12). In sea urchin eggs, poly(A) synthesis increases immediately after fertilization, and it was proposed that the length of the poly(A) in maternal mRNA may be related to its ability to function in protein synthesis (13)(14)(15).
Recently, the mRNAs of a variety of different eukaryotic cells and viruses were found to be methylated and to contain blocked 5'-terminal structures of the type, m'G( 5')ppp (5')N ' (16-24). These methylated sequences may play a role in mRNA biogenesis (24). In addition, 5'-terminal 7-methylguanosine in vesicular stomatitis virus (VSV), reovirus, and globin mRNAs is required for translation in wheat germ  (30)(31)(32), the postribosomal supernatant fraction (S105) of extracts of undeveloped embryos was used as a source of "undeveloped mRNA" (11). S105 was prepared according to Sierra et al. (27) with the exception that cysts were disintegrated in a French Dress according to Zasloff and Ochoa (33)  After incubation at 25" for 60 min, the amount of radioactivity incorporated into hot acid-insoluble material was determined as described (27). For measuring the time course of [asS]methionine incorporation, l-/11 samples from a total reaction volume of 12 ~1 were precipitated with 10% trichloroacetic acid containing 1% casamino acids and counted as described (34) (25,26). Translation of VW mRNAs in wheat germ extracts is also methylation-dependent (25). The methylated residue in mRNA essential for translation is the 7-methylguanosine in the 5'-terminal structure, m'G(Y)ppp(5')N (26). In wheat germ, this requirement is manifested at an early step in the initiation process of protein synthesis, at or prior to the level of binding of the 40 S ribosomal subunit to mRNA (39). To test whether there is a similar requirement for mRNA methylation in protein synthesis of lower eukaryotes, cellfree extracts containing 80 S ribosomes from undeveloped embryos and S105 supernatant from developing embryos of the crustacean, Artemia salinu (27), were programmed with methylated and unmethylated reovirus mRNA and with the homologous mRNA. Methylated reovirus mRNA stimulated polypeptide synthesis in these extracts, and the stimulation was unaffected by the addition of the methyl donor, S-adenosylmethionine, or its analog, S-adenosylhomocysteine, an inhibitor of methylation (Table I). Similar results were obtained with A. salina poly(A)-containing mRNA isolated from polysomes of developing embryos. Unmethylated reovirus mRNA was also translated in Artemia extracts, but to a lesser extent than methylated mRNA; again, addition of S-adenosylmethionine had little, if any, effect. However, in the presence of S-adenosylhomocysteine, polypeptide synthesis directed by unmethylated reovirus mRNA was inhibited by 80%. The results indicate that A. salina extracts can translate unmethylated viral mRNAs only after they are methylated, and suggest, as demonstrated below, that they contain an mRNAmethylating activity that converts unmethylated reovirus mRNA to the methylated form which is active in protein synthesis. Presumably, A. saliruz extracts also contain a level of methyl donor(s) sufficient to methylate the exogenous unmethylated viral mRNA because addition of S-adenosylmethionine to cell extracts had little effect on protein synthesis. 5'-Terminal 7-Methylguanosine in mRNA from Developed A. saline--As observed for methylated reovirus mRNA, the addition of S-adenosylmethionine or S-adenosylhomocysteine to A. salina extracts did not alter their ability to translate homologous mRNAs (Table I). These results suggested that mRNAs from developing embryos may already be methylated and contain 5'-terminal 'I-methylguanosine in a structure similar to that present in methylated reovirus mRNA, i.e. m'G(5')ppp (5')Gm.
This possibility was tested as follows. Poly(A)-containing mRNA from developing embryos was treated with periodate to oxidize free 2',3'-hydroxyl groups contained in 3'-terminal nucleotides and in presumptive 5'terminal structures (17) Most of the radioactivity (95%) stayed at the origin and was not a nucleotide derivative. It may correspond to a derivatized carbohydrate contaminant since high concentrations of trehalose and glycogen have been found in A. salina embryos (40). The remaining sH-labeled components containing 3% and 1% of the total SH radioactivity corresponded in mobility to adenosine and cytidine, respectively; presumably they were derived from the 3' ends of the mRNAs. The SH-labeled, P 1, and phosphatase-resistant anionic component was eluted and reanalyzed by descending paper chromatography (Fig. IA). More than 96% of the radioactivity chromatographed in one peak. This BH-labeled material was eluted and an aliquot digested with nucleotide pyrophosphatase followed by alkaline phosphatase was reanalyzed by chromatography.
The radioactivity was quantitatively recovered as the *H-labeled trialcohol derivative of 7-methylguanosine ( Fig. lB), which was identified further by paper chromatography.
The results indicate that the 5' termini of mRNA from developing embryos of A. salinn contain "capped" methylated structures similar to those in other viral and cellular mRNAs, i.e. 7-methylguanosine linked by an inverted 5'-5' pyrophosphate bridge to the rest of the RNA molecule.
In order to determine the number of phosphate groups in the cap structures, an aliquot of the [8H]borohydride-labeled RNA was digested with Penicillium nuclease and alkaline phosphatase and analyzed by paper chromatography. The cap material was recovered together with 8H-labeled nucleosides derived from the 3'-termini and analyzed by DEAE-cellulose column chromatography (17). As shown in Fig. 2, the nucleosides elute in the position of zero charge. The cap structures elute between the mononucleotide and dinucleotide optical density markers with a net charge of approximately -2.5. This value is consistent with the presence in the 5' termini of three phosphates, one partially neutralized by the positively  (17,26,37,38). One-half of the gH-labeled mRNA was digested with Penicillium nuclease and alkaline phosphatase and analyzed by paper electrophoresis at pH 3.5. The enzyme-resistant 5'-terminal material that migrated toward the anode near 5'-adenylic acid was eluted and reanalyzed (A) by descending paper chromatography in isobutyric acid/O.5 M NH,OH, 10/6, v/v. (B) the material indicated by the bracket in A was eluted, and one-half of the sample was digested with nucleotide pyrophosphatase and alkaline phosphatase and analyzed by paper electrophoresis at pH 3.5. m W' = trialcohol derivative of 7-methylguanosine; C = cytidine; A = adenosine.   (26). In order to determine if the 5'terminal 7-methylguanosine in A. salinu mRNA is required for translation, poly(A)-containing mRNA from polysomes of developing embryos was subjected to B elimination (37, 38). After treatment, its template activity was compared with control mRNA in cell-free protein-synthesizing extracts from A. salina and wheat germ (Table 11). Control mRNA, treated under the same conditions but without addition of periodate and aniline, was translated in both systems, although net synthesis of polypeptides was 3-fold greater in wheat germ extracts. Addition of S-adenosylmethionine or S-adenosylhomocysteine to either extract had little or no effect on translation of control mRNA, as observed with reovirus mRNAs (Table I) and methylated VW and globin mRNAs in wheat germ extracts (25, 26). After /3 elimination under conditions that removed 80 to 85% of the 5'-terminal 7methylguanosine from reovirus mRNA without chemical degradation (26), the translational capacity of A. salina mRNA was reduced by 67% and 72% in the heterologous and homologous systems, respectively (Table II). The loss of activity apparently is not due to internal cleavage of the mRNA because sedimentation profiles under denaturing conditions of reovirus and silk fibroin mRNAs were unchanged by @ elimination.*'* Furthermore, methylated reovirus mRNA which is inactivated by ,!? elimination can, in the presence of S-adenosylmethionine, stimulate protein synthesis in wheat germ extracts (26). This restoration of activity was shown to be due to the presence of some mRNA molecules with 5'-terminal ppG which were converted to 5'-m'GpppG.
Those molecules with 5'-terminal pppG" were not converted to active mRNA (26). A strict requirement for Y-terminal diphosphates by the vaccinia-derived guanylyltransferase has also been described recently (41). The results suggest that j3 elimination per se does not nonspecifically inactivate mRNA, and that 5'terminal 7-methylguanosine in A. salinu mRNA is required for translation in uitro.
5'-Terminal7-Methylguanosine in mRNA from Undeveloped Embryos-The results obtained with mRNA from developing embryos demonstrate that 5'-terminal 7-methylguanosine in mRNA is important for protein synthesis in brine shrimp. To determine whether the low level of protein synthesis in dormant cysts is due to an absence of 5'-terminal 7-methylguanosine in mRNA, the in uitro template activities of mRNA derived from undeveloped and developed embryos were comz Unpublished results.
*P. Gage, personal communication. pared. As previously found for reovirus unmethylated mRNA in wheat germ extracts (25), the translation of mRNA isolated from undeveloped embryos should be low in the presence of S-adenosylhomocysteine if 5'-terminal 7-methylguanosine is absent from these mRNAs. However, as shown in Fig. 3, the kinetics of incorporation of ["Slmethionine into acid-precipitable material directed by mRNA preparations from both developed and undeveloped embryos was unaffected by Sadenosylhomocysteine, although the activity of the former mRNA was 2-to 3-fold greater.
The findings in Fig. 3 suggest that mRNA isolated from undeveloped embryos also contains 5'-terminal 7-methylguanosine. Poly(A)-containing mRNA prepared from dry cysts was oxidized with periodate, reduced with [BH]borohydride, digested with Penicillium nuclease and alkaline phosphatase, and analyzed by paper electrophoresis at pH 3.5 as in Fig. 1. A single peak of aH-labeled anionic material was obtained, and it was further purified by paper chromatography (Fig. 4A). The predominant *H-labeled component (Fractions 22 and 23) was eluted, an aliquot digested with nucleotide pyrophosphatase and alkaline phosphatase, and reanalyzed by electrophoresis. More than 85% of the *H was obtained as the trialcohol derivative of 'I-methylguanosine (Fig. 4B) Reaction mixtures containing no exogenous viral mRNA were treated identically. The incubation mixtures were extracted with phenol, and the RNA was recovered by alcohol precipitation, digested with P, nuclease followed by alkaline phosphatase and analyzed by paper electrophoresis at pH 3.5. As shown in Fig. 5, a predominant [8H]methyl-labeled component migrating in the position of pG was obtained only from the digest of RNA isolated from the reaction mixture to which exogenous unmethylated reovirus mRNA was added (Fig. 5A). The aH-labeled material in this peak was eluted and analyzed by paper chromatography. Two components were resolved, a minor one which migrated in the position of marker m'GpppGm obtained from the 5'termini of reovirus mRNA, and a major one which migrated more slowly in the position of m'GpppG (Fig. 5B). The latter marker compound was prepared from reovirus mRNA methylated in wheat germ extract (26). Each component was eluted, treated with nucleotide pyrophosphatase and alkaline phosphatase, and reanalyzed by paper electrophoresis at pH 3.5. The *H-labeled component that migrated with the authentic m'GpppG'" yielded two equal peaks of radioactivity, one which migrated with 7-methylguanosine, and another which was close to the origin, the position of 2'-O-methylguanosine (Fig. SD) Unmethylated reovirus mRNA (40 pg) synthesized in vitro as described previously (34) was incubated for 10 min in an extract containing SlO5 supernatant fraction from developed A. salinu and ribosomes from undeveloped embryos in the presence of [*H]methyl-S-adenosylmethionine as described under "Materials and Methods." An identical control incubation mixture contained no added viral mRNA. RNA was recovered from the incubation mixtures by phenol extraction and alcohol precipitation and digested with Penicillium nuclease, followed by alkaline phosphatase. A, paper electrophoresis of the enzyme digests with (---) and without (---) reovirus mRNA. The profiles from separate analyses are superimposed. B, paper chromatography in isobutyric acid/NH,OH of the material eluted from the region indicated by the bracket in A. C, the material in the region of the marker m'GpppG in B was eluted, digested with nucleotide pyrophosphatase and alkaline phosphatase, and analyzed by paper electrophoresis at pH 3.5. D, material in the region of the marker m'GpppG" in B was analyzed as described for Part C. G = guanosine; U = uridine; PC, PA, pG, pU = 5'-monophosphates of cytidine, adenosine, guanosine, and uridine, respectively. used, the predominant methylation of reovirus mRNA occurred specifically at position N' of the 5'-terminal guanosine. The mRNA methylase activity was absent from 0.5 M KCl-washed ribosomes, and was recovered in the high speed supernatant fraction (data not shown), consistent with a nonribosomal localization, as in wheat germ extracts (39).
The same procedures were used to determine whether mRNA-methylating activities are also present in undeveloped A. salinu. The results were very similar to those obtained with SlO5 supernatant fraction from developed embryos, with the exception that lesser amounts of P'-0-methylated 5'terminal structures were observed in the mRNA methylated by S105 from undeveloped embryos (data not shown). The findings indicate that similar, possibly identical, mRNAmethylating activities are present in developed and undeveloped A. salina embryos, and that both convert reovirus unmethylated mRNA to molecules containing 5'-terminal m7GpppGm or m'GpppG. DISCUSSION Undeveloped, encysted embryos of A. salina contain little, if any, polysomes, and cell-free extracts prepared from them are almost inactive in protein synthesis (27,(30)(31)(32). However,