Sequence of methylated nucleotides at the 5''-terminus of adenovirus-specific RNA.

RNA labeled with [methyl-3H] methionine and [14C]uridine was isolated from the cytoplasm of adenovirus-infected cells and purified by poly(U)-Sepharose chromatography and hybridization to filters containing immobilized adeovirus DNA. Analysis by dimethyl sulfoxide-sucrose gradient sedimentation suggested that the major mRNA species were methylated. 7-Methylguanosine was identified at the 5'-terminus of the advenovirus-specific RNA and could be removed by periodate oxidation and beta-elimination. Structures of the type m7G(5')ppp(5')Nm containing the unusual nucleoside N6, O2'-dimethyladenosine, and smaller amounts of 2'-O-methyladenosine were isolated by DEAE-cellulose chromatography after P1 nuclease digestion of the RNA. Evidence for some 5'-terminal sequences, m7G(5')ppp(5')m6AmpNm, with additional 2'-O-methylribonucleosides was also obtained. A base-methylated nucleoside, N6-methyladenosine, is located within the RNA chain and is released as a mononucleotide by alkali hydrolysis.     

"Cap" sequences in poly(A)-rich RNA from dry wheat embryos

Although template-active RNA in dry seeds and embryos has attracted widespread interest, there have been no published reports about 5'-terminal "capping" sequences in such RNA. Boro[3H]hydride labeling of periodate-oxidized termini and high performance liquid chromatography of cap oligonucleotides have been used to compare terminal sequences in poly(A)-rich RNA from dry and germinating embryos. As is the case in germinating embryos, poly(A)-rich RNA from dry embryos contains only "type 0" cap sequences, i.e., m7G(5')ppp(5')N, in which m7G is the 7-methylguanosine cap and N is any of the classical ribonucleosides: adenosine (A), guanosine (G), cytidine (C),a nd uridine (U). Striking differences between the cell-free translational capacities of bulk messenger RNA (mRNA) populations from dry and germinating embryos are not reflected in signal differences in their proportions of "type 0" cap structures: in general, there is approximately 40% m7G(5')ppp(5')A, with roughly equivalent amounts of m7G(5')ppp(5')G and m7G(5')ppp(5')C accounting for most of the remaining sequences. The findings with mRNA from dry plant embryos serve to emphasize interesting differences between patterns of methylation in the capped and uncapped RNA molecules in higher plants and animals; the differences have not been previously noted in the literature and are the subject of brief comment in this paper.     

5'-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA.

The 5'-terminal oligonucleotides m7G(5')ppp(5')NmpNp and m7G(5')ppp(5')NmpNmpNp were isolated by DEAE-cellulose column chromatography after enzymatic digestion of 32P- or methyl-3H-labeled poly(A)" HeLa cell mRNA. The recovery of such oligonucleotides indicated that a high percentage of mRNA has blocked termini. The dimethylated nucleoside, N6, O2'-dimethyladenosine (m6Am), as well as the four common 2'-O-methylribonucleosides (Gm, Am, Um, Cm) were present in the second position linked through the triphosphate bridge to 7-methylguanosine (m7G) whereas little m6Am was in the third position. The only internal methylated nucleoside, N6-methyladenosine (m6A), was found exclusively as m6ApC and Apm6ApC after digestion with RNase A, T1, and alkaline phosphatase. Digestion with RNase A and alkaline phat pyrimidines are present in much smaller amounts or absent from this position. These results imply a considerable sequence specificity since there are thousands of different mRNA species in HeLa cells. Our studies are consistent with the following model of HeLa cell mRNA in which Nm may be m6Am, Gm, Cm, Um, or Am and one or more m6A residues are present at an unspecified internal location: m7G(5')ppp(5')Nm-(Nm)---(G or A)-m6A-C---(A)100-200A.     

The methylation state of poly A-containing messenger RNA from cultured hamster cells.

The poly A-containing mRNA of cultured hamster (BHK-21) cells has been examined with regard to methylation status. Steady state-labeled mRNA was obtained by incubating cells for 20-22h in the presence of [methyl-3H]-methionine and 32Pi. The degree of methylation of this RNA was 1.8 methyl groups per 1000 nucleotides, or 4-5 methyl groups on the average per molecule. The nature of the methylated residues was determined by paper chromatography and electrophoresis of acid and alkaline hydrolysates, by DEAE cellulose chromatography of alkaline hydrolysates and of T2 RNase digests, and by examining the effect of subjecting samples to "beta-elimination." Approx. half of the methyl groups occurred in standard ("internal") linkage, 10% as m5Cp and 40% as m6Ap residues. The remainder occurred at least for the most part in "blocked" 5'-termini with the presumptive structure m7G(5')ppp(Nm)p.., where Nm was Gm, m6Am, Um, or Cm.     

The 5' terminal structure of the methylated mRNA synthesized in vitro by vesicular stomatitis virus.

The 5' terminal structure of the mRNA synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus in the presence of S-adenosyl-L-methione consists of 7-methyl guanosine linked to 2'-O-methyl adenosine through a 5'-5' pyrophosphate bond as m7G(5')ppp(5')A-m-p ... The alpha and beta phosphated of GTP and alpha phosphate of ATP are incorporated into the blocked 5' terminal structure.     

Characterization of 5'-terminal methylation of human alpha- and beta-globin mRNAs

After incubating peripheral blood of various anemic individuals with [methyl-3H]methionine, 5'-terminal cap structures and the sequential methylation of human globin mRNAs were determined. Human alpha- and beta-globin mRNAs have the same methylated nucleotides at their 5' ends as the alpha- and beta-globin mRNAs of mouse and rabbit: m7G(5')ppp(5')[m6Am/Am]pCmp. The order of methyl group addition to human globin mRNA is also similar to that of mouse globin mRNA in that methylation of the 5'-terminal guanosine and the ribose of adenosine occurs earlier than that of the base of adenosine and the ribose of cytidine. Thus, both the cap structures and the order of methylation are conserved in the globin mRNAs of these species, suggesting that variation in the structure of the 5' terminus of globin mRNAs may alter the function of these molecules.     

Impairment of reovirus mRNA 'cap' methylation in interferon-treated mouse L929 cells.

Reovirus mRNAs synthesized in vitro by the virionassociated enzyme have a 5' 'cap 1' structure (m7G(5')ppp(5')GmpCp...). However, about one third to one half of the reovirus mRNAs formed in mouse L929 cells have a 5' 'cap 2' structure (m7G(5')ppp(5')GmpCmp...) and the rest have a 5' 'cap 1' structure. The finding that virus mRNA 'cap' methylation is impaired in extracts of interferon-treated cells prompted us to study the effect of interferon on virus mRNA 'cap' methylation in vivo. Using labeling with [3H]-guanosine and dual labeling with [3H]methionine and [14C]uridine we compared the 5' structures of reovirus mRNAs accumulating between 5 and 11 h after infection in: L929 cells treated with 390 to 2600 U/ml of a partially purified mouse interferon preparation and untreated L929 cells. The treatment resulted in a 70 to 98% decrease in the 24 h virus yield and in a 50 to 55% decrease in the label accumulated in virus mRNAs. The 'capping' of virus mRNAs and the methylation of their 5' terminal and adjacent G residues were not diminished in interferon-treated cells. However, the percent of 'cap 2' termini was 36 to 47% lower in virus mRNAs from interferon-treated cells than in virus mRNAs from control cells. The interferon treatment did not result in the appearance of additional methylated nucleotides in the virus mRNAs.     

Methylated constituents of Aedes albopictus poly (A)-containing messenger RNA.

Poly (A)-containing mRNA prepared from cultured mosquito (Aedes albopictus) cells was found to contain methylated 5'-terminal "caps" as well as internal m6A residues. Both type I [m7G(5')ppp(5')Xmp] and type II [m7G(5')ppp(5')XmpYmp] caps were present, at molar ratio of ca five to one. All four common RNA bases were represented in the second position (Xm) of the caps, adenine being the most abundant and N6-methyladenine being absent. The four bases were also represented in the third position (Ym), but here uracil was the predominant base. There was approximately one internal m6A residue for every three caps. These studies demonstrate that mRNA from an invertebrate source can have a methylation pattern comparable with that of mammalian cells in it complexity.     

mRNA methylation and protein synthesis in extracts from embryos of brine shrimp, Artemia salina.

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.     

Multiple methylated cap sequences in adenovirus type 2 early mRNA.

The methylated constituents of early adenovirus 2 mRNA were studied. RNA was isolated from polyribosomes of cells double labeled with [methyl-3H]methionine and 32PO4 from 2 to 7 g postinfection in the presence of cycloheximide. Cycloheximide ensures that methylation and processing are performed by preexisting host cell enzymes. RNA was fractionated into polyadenylic [poly(A)]+ and poly(A)- molecules using poly(U)-Sepharose, and undergraded virus-specific RNA was isolated by hybridization to viral DNA in 50% formamide at 37 degrees C. Viral mRNA was digested with RNase T2 and chromatographed on DEAE-Sephadex in 7 M urea. Two 3H-labeled RNase T2-resistant oligonucleotide fractions with charges between -5 and -6 were obtained, consistent with two classes of 5' terminal methyl "cap" structures, m7G(5')ppp(5')NmpNp (cap 1) and m7G(5')ppp(5')NmNmpNp (cap 2) (Nm is a ribose 2'-O-methylation). The putative cap 1 contains all the methylated constituents of cap 1 plus Cm. The molar ratios of m7G to 2'-O-methylnucleosides is about 1.0 for cap 1 and 0.5 for cap 2, consistent with the proposed cap structures. Most significant, compositional analysis indicates four different cap 1 structures and at least three different cap 2 structures. Thus there is a minimum of seven early viral mRNA species with different cap structures, unless each type of mRNA can have more than one 5' terminus. In addition to methylated caps, early mRNA contains internal base methylations, exclusively as m6A, as shown by analyses of the mononucleotide (-2 charge) fraction. m6A was present in the ratio of 1 mol of m6Ap per 450 nucleotides. Thus viral mRNA molecules contain two to three internal m6A residues per methyl cap, since there is on the average 1 cap per 1,250 nucleotides.     

Detection in HeLa cell extracts of a 7-methyl guanosine specific enzyme activity that cleaves m7GpppNm.

Extracts prepared from HeLa cells contain an enzymatic activity which cleaves m7G(5')ppp(5')Gm to m7pG and ppGm. The activity exhibits a high degree of substrate specificity and does not cleave G(5')ppp(5')G or the ring opened derivative of m7GpppGm which has lost the positive charge from the N7 position of m7G. m7GpppGm as the 5' terminal structure of intact reovirus mRNA is resistant to attack by the pyrophosphatase activity, but becomes partially sensitive in the 5' terminal fragment consisting of 7-10 nucleotides derived from the same mRNA by T1 RNAase digestion. m7G(5')ppp(5')GmpCp is completely sensitive to cleavage resulting in the release of m7pG without generation of m7GpppGm as an intermediate. These results establish the existence of a 7-methyl guanosine specific pyrophosphatase activity in HeLa cells.     

Relative importance of 7-methylguanosine in ribosome binding and translation of vesicular stomatitis virus mRNA in wheat germ and reticulocyte cell-free systems.

Vesicular stomatitis virus mRNAs with these four types of 5'-termini, (a) m7G5'ppp5'(m)Am, (b) ppp5'(m)Am, (c) m7G5'-ppp5' Am, and (d) G5'ppp5'A, were prepared and their translation and ribosome binding analyzed in wheat germ and reticulocyte cell-free protein synthesis systems. The relative efficiencies of translation of individual vesicular stomatitis virus (VSV) mRNAs having type 2 termini ranged from 23 to 29% of the control (type 1) RNA in the reticulocyte system and 6 to 7% of control RNA in the wheat germ system. A similar difference between the two systems was seen in ribosome-binding experiments in which type 2 RNA formed an 80 S initiation complex with high efficiency (70% of control type 1 RNA) in the reticulocyte system, but with low efficiency (17% of control RNA) in the wheat germ system. Similar differences in the importance of m7G in translation in the two systems were seen when VSV mRNAs synthesized in vitro with type 3 and type 4 termini were analyzed. However, the analysis of type 4 RNA (which was synthesized in vitro in the presence of S-adenosylhomocysteine) was complicated by the presence of abnormally large poly(A) at its 3'-end. Another series of experiments showed that compounds such as 5'pm7G and m7G5'ppp5'Np are potent and specific inhibitors of translation of all types of VSV mRNAs in the wheat germ system (greater than 98% inhibition) but cause less than 20% inhibition of translation in the reticulocyte system. Taken together, all of the results indicate that a 5'-terminal m7G is far more important in translation of VSV mRNAs in the heterologous plant cell-free system than in the reticulocyte lysate system.     

Two methyltransferase activities in the purified virions of vesicular stomatitis virus.

In addition to an RNA-dependent RNA polymerase, purified vesicular stomatitis virus contains a methyltransferase activity which transfers the methyl group from the methyl donor, S-adenosyl-L-methionine, to two positions in the 5'-terminal capped structure of the nascent mRNA's synthesized in vitro as 7mG-(5)'ppp(5')Apm... In the present study it is shown that two distinct methyltransferase activities are discernible in the purified virus. The in vitro concentrations of the methyl donor specify the number and location of the methyl groups transferred to the capped 5'-termini of VSV mRNA's. Limited concentrations of the methyl donor result in a single methylation of the penultimate base in the 2'-hydroxyl position, that is, G(5')ppp(5')Apm..., whereas saturating concentrations of the methyl donor methylate the blocking guanosine residue at the 7-position, resulting in the dimethylated cap, 7mG(5')ppp(5')Apm... Pulse-chase experiments demonstrate that the monomethylated cap structure is the precursor substrate for the dimethylated cap. In this respect, vesicular stomatitis virus system is quite distinct from the vaccinia and reovirus systems. Virus purified from different host cells including hamster, mouse, and human contain both methyltransferase activities. The mRNA's containing monomethylated capped structures are poor templates for protein synthesis in vitro.     

5''-Terminal and internal methylated nucleosides in herpes simplex virus type 1 mRNA.

RNA labeled with [methyl-3H]methionine and/or [32P]orthophosphate was isolated from the polyribosomes of herpes simplex virus (HSV) types 1-infected cells and separated into polyadenylylated [poly(A+)]and non-polyadenylylated [poly(A-)] fractions. Virus-specific RNA was obtained by hybridization in liquid to either excess HSV DNA or filters containing immobilized HSV DNA. Analysis in denaturing sucrose gradients indicated that HSV-specific poly(A+) RNA sedimented in a broad peak, with a modal S value of 20. The ratio of [3H]methyl to 32P decreased with increasing size of RNA, suggesting that each RNA chain contains a similar sumber of methyl groups. Further analysis indicated an average of one RNase-resistant structure of the type m7G(5')pppNmpNp or m7G(5')pppNmpNmpNp per 2,780 nucleotides. The following components were identified in the 5'-terminal oligonucleotides of polyribosome-associated HSV-specific poly(A+) and poly(A-) RNA: 7-methylguanosine, N6,2'-O-dimethyladenosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and denosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and cytidine. The most common 5'-terminal sequences were m7G(5')pppm6Am and m7G(5')pppGm. An additional modified nucleoside, N6-methyladenosine, was present in an internal position of HSV-specific RNA.