Sequence specificity of internal methylation in B77 avian sarcoma virus RNA subunits.

Following ribonuclease digestion of methyl-3H-labeled B77 avian sarcoma virus RNA subunits, methylated oligonucleotides were isolated by diethylaminoethylcellulose chromotogrpahy. Partial nucleotide sequences were deduced from the known enzymatic specificities of the ribonucleases. In addition to methylated nucleosides in the 5'-terminal cap structure, m7G(5')GmpCp, N6-methyladenosine(m6A) was found to be present in only two internal sequences of the RNA molecule, Gpm6ApC and Apm6ApC. The average numbers of methylated nucleosides per RNA subunit are about 12-13 in Gpm6ApC, 1-2 in Apm6ApC, and 2 in m7GpppGmpCp. The sequences containing m6A in B77 sarcoma virus RNA are identical to m6A-containing sequences previously reported for the bulk mRNA from HeLa cells (Wei, C.M., Gershowitz, A., and Moss, B. (1976), Biochemistry 15, 397-401). Analysis of the oligonucleotides produced by RNase A digestion indicated that the sequence of bases on the 5' side of these trinucleotides is not specific. The oligonucleotide profile, however, was highly reproducible in different virus preparations. This suggests that the methylations occur at specific positions on the RNA molecule. Some of the methylated oligonucleotides produced by RNase A digestion appear to be present in less than molar amounts. Several hypotheses are proposed to explain this result.     

Capping structures of simian virus 40 19S and 16S mRNAs.

In vivo [methyl 3H]-labeled SV40 19S and 16S mRNA species were purified and their internal methylation as well as their capping structures analyzed. SV40 viral mRNA sedimenting in the 19S region contains approximately equal proportions of m7GpppAm and m7Gppm6Am, while the 16S mRNA contains mainly m7Gpppm6Am. N6 methyl adenosine is located internally within the RNA chains of both the 19S and 16S species.     

Electron microscopic evidence for splicing of SV40 late mRNAs.

Poly(A)-containing SV40 cytoplasmic RNA was hybridized with linear double-stranded SV40 DNA and formed RNA displacement loops (R loops). The structures visualized in the electron microscope are consistent with the conclusion that the leader sequences at the 5' ends of the 16S and 19S late mRNAs are not coded immediately adjacent to the main portions of the mRNAs. These data are consistent with either segmentation of the leaders or heterogeneity of their lengths. Measurements carried out on the R loop structures have provided the locations, on the physical map of SV40 DNA, for the bodies and leaders of the 16S and 19S late mRNAs, and the lengths of the bodies, leaders and the corresponding intervening DNA sequences.     

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.     

Polysomes and polysomal mRNAs in SV40-infected CV-1 cells. In vivo observations.

SV40-specific polysomes are relatively larger than host polysomes. Both 16 S and 19 S virus-specific late mRNAs are found associated with these polysomes and they are present in a ratio of approximately 2 : 1. The rates at which these virus-specific mRNAs are translated are the same, so that the relative amounts of virus-specific polypeptides synthesized in the virus-infected cells is determined by the relative amounts of 16 S and 19 S mRNAs coding for them.     

Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing.

N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus. Specific fragments of 32P-labeled Rous sarcoma virus RNA were isolated by hybridization with complementary DNA restriction fragments spanning nucleotides 6185 to 8050. RNA was digested with RNase and finger-printed, and individual oligonucleotides were analyzed for the presence of m6A by paper electrophoresis and thin-layer chromatography. With this technique, seven sites of methylation in this region of the Rous sarcoma virus genome were localized at nucleotides 6394, 6447, 6507, 6718, 7414, 7424, and 8014. Further, m6A was observed at two additional sites whose nucleotide assignments remain ambiguous. A clustering of two or more m6A residues was seen at three positions within the RNA analyzed. Modification at certain sites was found to be heterogeneous, in that different molecules of RNA appeared to be methylated differently. Previous studies have determined that methylation occurs only in the sequences Gm6AC and Am6AC. We observed a high frequency of methylation at PuGm6ACU sequences. The possible involvement of m6A in RNA splicing events is discussed.     

Location of the sequences coding for capsid proteins VP1 and VP2 on polyoma virus DNA.

The 19S and 16S polyoma virus late mRNAs have been separated on sucrose-formamide density gradients and translated in vitro. The 16S RNA codes only for polyoma capsid protein VP1, while the 19S RNA codes in addition for capsid protein VP2. Since the 19S and 16S species have been previously mapped on the viral genome, these results allow us to deduce the location of the sequences coding for VP1 and VP2. Comparison of the chain lengths of the capsid proteins with the size of the viral mRNAs coding for them suggests that VP1 and VP2 are entirely virus-coded. Purified polyoma 19S RNA directs the synthesis of very little VP1 in vitro, although it contains all the sequences required to code for the protein. The initiation site for VP1 synthesis which is located at an internal position on the messenger is probably inactive either because it is inaccessible or because it lacks an adjacent "capped" 5' terminus. Similar inactive internal initiation sites have been reported for other eucarotic viral mRNAs (for example, Semliki forest virus, Brome mosaic virus, and tobacco mosaic virus), suggesting that while eucaryotic mRNAs may have more than one initiation site for protein synthesis, only those sites nearer the 5' terminus of the mRNA are active.     

Characterization of Two Simian Virus 40-Specific RNA Molecules from Infected BS-C-1 Cells

Two discrete simian virus 40 (SV40) RNA species sedimenting at 19 and 16S, respectively, that are present in infected BS-C-1 cells were characterized with respect to the base composition and the ribonuclease T1 fingerprints. The base composition of the 19S SV40 RNA was found to be cytidylic acid (C), 23.0; adenylic acid (A), 28.3; guanylic acid (G), 23.9; and uridylic acid (U), 24.8; that of the 16S SV40 RNA was C, 19.3; A, 34.0; G, 22.0; and U, 24.7 mol%. Analysis of the ribonuclease T1 fingerprints indicated a difference in the base sequence of the 19 and 16S SV40 RNA. The presence of long sequences of adenylic acid residues (poly A) in these viral RNAs was confirmed.     

Mechanism for suppression mRNA translation with antisense oligonucleotides

Experimental studies of the effects of antisense oligonucleotides on translation of mRNAs in cell-free systems are reviewed. Oligonucleotides complementary to the leader sequences or to the sequence overlapping the initiating codon region of mRNAs inhibit translation of the messengers. In the presence of ribonuclease H, oligodeoxyribonucleotides and their phosphorothioate analogs complementary either to the mentioned mRNA regions or to the mRNA coding sequence suppress the translation due to the RNAs cleavage. This inhibition-enhancing mechanism does not operate in the case of the oligonucleotide analogs--oligonucleoside methylphosphonates and oligonucleotides built of the alpha-nucleosides, since the complexes formed by RNA and these analogs are not substrates of the ribonuclease H. The translation inhibition efficiency is determined by the oligonucleotides lengths and by the availability of the complementary sequence in the mRNA structure. The oligonucleotides inhibitory power can be improved by the coupling to the oligonucleotides of the intercalating groups and the reactive groups.     

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.     

Heterogeneity of the 5' terminus of hen ovalbumin messenger ribonucleic acid.

The 5'-terminal sequence of hen ovalbumin mRNA was investigated using a novel labeling method. Ovalbumin mRNA was purified by hybridization to complementary DNA coupled to cellulose. The mRNA thus purified was shown to be 97.9% pure by hybridization with plasmid DNA containing sequences to the messengers coding for conalbumin and ovomucoid, the next two most abundant messengers of oviduct. After digestion with RNase T1 and alkaline phosphatase, 5'-terminal capped oligonucleotides were selected by binding to anti-m7G-Sepharose. These were then labeled using RNA ligase and [5'-32P]pCp, separated by two-dimensional gel electrophoresis, and sequenced by partial digestion with base-specific ribonucleases. A nested set of three capped oligonucleotides was identified. Their structures and relative abundances were m7GpppAUACAG, 3% m7GpppACAUACAG, 61+; and m7GpppGUACAUACAG, 36%.