Heterogeneity of the 5'' terminus of late mRNA induced by a viable simian virus 40 deletion mutant.

d1-1811 is a viable simian virus 40 deletion mutant which lacks the DNA region corresponding to the major capping site of the late viral RNA. The exact size of the deletion (40 base pairs) was determined by comparison of the mutant DNA sequence with the wild-type simian virus 40 (strain 776) DNA sequence. Although d1-1811 forms somewhat smaller plaques, the amount of viral RNA late after infection was not significantly reduced compared with that of the wild type. Virus-specific, polyadenylate-containing, 32P-labeled late RNA was purified from the cytoplasm and enzymatically degraded to characterize the 5' terminus. The cap-containing oligonucleotides were isolated, and their structures were analyzed by further digestion. Instead of a single cap structure, we found a variety of capped 5' termini, with adenosine caps occurring much more frequently than guanosine caps. Nevertheless, there was a remarkable homology between both types of terminal sequences. Conceivably, the minor cap population present in wild-type simian virus 40 late mRNA may correspond to the collection of capped termini identified in the d1-1811 late mRNA . Cellular cytoplasmic RNA shows a similar pattern of cap structures, but the relative abundance is quite different.     

At least 104 nucleotides are transposed from the 5' terminus of the avian sarcoma virus genome to the 5' termini of smaller viral mRNAs.

Cells producing avian sarcoma virus (ASV) contain at least three virus-specific mRNAs, two of which are encoded within the 3' half of the viral genome. Each of these viral RNAs can hybridize with single-stranded DNA(cDNA5') that is complementary to a sequence of 101 nucleotides found at the 5' terminus of the ASV genome, but not within the 3' half of the genome. We proposed previously (Weiss, Varmus and Bishop, 1977) that this nucleotide sequence may be transposed to the 5' termini of viral mRNAs during the genesis of these RNAs. We now substantiate this proposal by reporting the isolation and chemical characterization of the nucleotide sequences complementary to cDNA5' in the genome and mRNAs of the Prague B strain of ASV. We isolated the three identified classes of ASVmRNA (38, 28 and 21S) by molecular hybridization; each class of RNA contained a "capped" oligonucleotide identical to that found at the 5' terminus of the ASV genome. When hybridized with cDNA5', each class of RNA gave rise to RNAase-resistant duplex hybrids that probably encompassed the full extent of cDNA5'. The molar yields of duplex conformed approximately to the number of virus-specific RNA molecules in the initial samples; hence most if not all of the molecules of virus-specific RNA could give rise to the duplexes. The duplexes prepared from the various RNAs all contained the capped oligonucleotide found at the 5' terminus of the viral genome and had identical "fingerprints" when analyzed by two-dimensional fractionation following hydrolysis with RNAase T1. In contrast, RNA representing the 3' half of the ASV genome did not form hybrids with cDNA5'. We conclude that a sequence of more than 100 nucleotides is transposed from the 5' end of the ASV genome to the 5' termini of smaller viral RNAs during the genesis of these RNAs. Transposition of nucleotide sequences during the production of mRNA has now been described for three families of animal viruses and may be a common feature of mRNA biogenesis in eucaryotic cells. The mechanism of transposition, however, and the function of the transposed sequences are not known.     

Polyoma virus early and late mRNAs in productively infected mouse 3T6 cells.

We mapped polyoma virus-specific mRNAs isolated from productively infected mouse 3T6 cells on the viral genome by analyzing nuclease S1-resistant RNA-DNA hybrids. The polyoma early mRNAs, which code for the three T antigens, have several 5' ends near 73 map units (m.u.). During the late phase of infection an additional 5' end is found near 71 m.u. All of the major early mRNAs have common 3' ends at 26.01 m.u. There is a minor species of early mRNA with a 3' end at 99.05 m.u. There are two proximal and two distal splice junctions in the early region which are used to generate three different spliced early mRNAs. There are three late mRNAs encoding the three virion proteins, VP1, VP2, and VP3. The late mRNAs have common 3' ends at 25.34 m.u. The late mRNAs have heterogeneous 5' leader sequences derived from the region between 65.53 and 68.42 m.u. The leader sequences are joined to the bodies of the messages coding for VP2, VP3, and VP1 at 66.59, 59.62, and 48.57 m.u., respectively. These results confirm and extend previous analyses of the fine structure of polyoma mRNAs.     

Distinct RNA elements confer specificity to flavivirus RNA cap methylation events

The 5' end of the flavivirus plus-sense RNA genome contains a type 1 cap (m(7)GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5' terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2'-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5' stem-loop structure; in contrast, 2'-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5' viral RNA of 20 nucleotides. The cap analogues GpppA and m(7)GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m(7)Gppp-terminated RNAs suggest that the 5' termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.     

Kinetics of Novikoff cytoplasmic messenger RNA methylation.

Methylation patterns of Novikoff cytoplasmic mRNA were determined as a function of labeling time with L-[methyl-3H]methionine. The 5'-terminal m7G could be released from whole mRNA by treatment with nucleotide pyrophosphatase. Subsequent alkaline phosphatase treatment of this mRNA, followed by KOH digestion, yielded N'mpNp and N'mpNp from cap 1 (m7GpppN'mpN) and cap 2 (m7GpppN'mpN'mpN), respectively. Our results indicate that the relative amounts of labeled cap structures do change with time and that the amount of internal N6-methyladenosine decreases, relative to 5'-cap structures, as the cytoplasmic mRNAs age and the average size decreases. The formation of cap-2 structures by the addition of second 2'-O-methyl group at position N'm appears to be cytoplasmic event. Thus, after very short labeling times, greater than 80% of the labeled methyl groups in cap 2 are found in this position. These results, along with earlier data obtained on L-cell heterogeneous nuclear RNA methylation, are consistent with a model in which the nucleus is the cellular site of three mRNA methylation events producing 5'-terminal m7G, the first 2'-O-methylnucleoside (N'm) found in cap-1 structures and internal N6-methyladenosine. Subsequently, these nuclear methylations are followed by the cytoplasmic methylation at N'm. Analysis of the methynucleoside composition of cap-1 structures, along with comparable "core" structures (m7GpppN'm) generated from cap-2 by removal of N'm, indicates that at any single labeling time the methylnucleoside composition of a given cap-1 and the cap-2 "core" structure is remarkably similar. On the other hand, comparisons of the methylnucleoside composition of the cap structures at different labeling times indicate an increase in Cm in the first 2'-O-methylnucleoside (N'm) with time.     

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.     

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.     

"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.     

Antigody-nucleic acid complexes. Inhibition of translation of silkmoth chorion messenger ribonucleic acid with antibodies specific for 7-methylguanosine.

Antibodies specific for 7-methylguanosine (m7G) were evaluated for their ability to inhibit the translation of chorion mRNA in a wheat germ, cell-free amino acid incorporating system. Results obtained with antibody concentrations of 0.5--1.5 microM revealed dose-dependent inhibition of [3H]-labeled amino acid incorporation into acid-insoluble radioactivity. Inhibition of translation was attributed to the interaction of anti-m7G antibodies with the 5' termini of chorion mRNAs on the basis that (a) anti-m7G antibodies coupled to Sepharose (anti-m7G-Sepharose) immunospecifically retained 5'-terminal cap structures of chorion mRNAs, i.e., m7G (5')ppp(5')Nm, (b) significant inhibition of translation required a 2-h preincubation of anti-m7G antibodies with mRNA, and (c) similar preincubation periods with anti-m7G antibodies in the presence of the competing nucleoside hapten (m7G) obviated the inhibitory effect of the antibody. The nature of the anti-m7G antibody-mRNA complex was examined by digesting chorion mRNA with nuclease P1 before (predigested) and after (postdigested) immunospecific adsorption to anti-m7G-Sepharose adsorbent. Whereas predigested preparations yielded a single cap structure of the type m7G(5')ppp(5')N, the predominating cap in the postdigested sample was m7G(5')ppp(5')NpNpN. These latter data revealed that the nucleotide sequence adjacent to the cap was not significantly masked by the antibody and suggest the utility of anti-m7G antibody as a site-specific probe.