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PubMed1974

Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells.

The poly(A) tract found in eukaryotic mRNA was used to study methylation in mRNA obtained from Novikoff hepatoma cells. Methyl labeling of RNA was achieved with L-[methyl-(3)H]methionine under conditions that suppress radioactive incorporation into the purine ring. RNA that contains a poly(A) segment was obtained from polysomal RNA by chromatography on oligo(dT)-cellulose. Sucrose density gradient centrifugation of this RNA revealed a pattern expected for mRNA. The composition of the methyl-labeled nucleosides in the RNA was analyzed after complete enzymatic degradation to nucleosides. By use of DEAE-cellulose (borate) chromatography, which separates 2'-O-methylnucleosides from normal and base-methylated nucleosides, about 50% of the radioactivity was recovered in the 2'-O-methylnucleoside fraction and 50% in the base-methylnucleoside fraction. High-speed liquid chromatography (Aminex A-5) of the 2'-O-methylnucleoside fraction produced four peaks coincident with the four 2'-O-methylnucleoside standards. Analysis of the base-methylnucleoside fraction revealed a unique pattern. While ribosomal RNA and tRNA possessed complex base-methylnucleoside patterns, the distribution in mRNA was quite simple, consisting predominantly of N(6)-methyladenosine. These results demonstrate a unique distribution of methylated nucleosides in mRNA. By analogy to ribosomal RNA synthesis, the presence of methylnucleosides in mRNA may reflect a cellular mechanism for the selective processing of certain mRNA sequences.

Adenosine↗
PubMed1974

Localization of adenovirus 2 messenger RNA's to segments of the viral genome defined by endonuclease R-R1.

Adenovirus 2 mRNAs synthesized in productive infection were assigned to specific regions of the genome by hybridization to unique fragments of viral DNA. Radioactive viral RNA synthesized early or late in infection was first fractionated by polyacrylamide gel electrophoresis. Eluted RNAs were then tested for complementary hybrid formation with each of the six fragments of adenovirus 2 DNA generated by cleavage with endonuclease R.R1. Early RNA species migrating as 13S, 19S, and 20S RNAs were identified as transcription products of fragments A, B, and D, respectively. In addition to the 13S RNA transcribed from A fragment DNA, 13S RNA also hybridized to the D and E fragment DNAs; 11S RNA annealed to both A and B fragments. The RNA that hybridized to fragment C DNA was heterogeneous in size. Viral RNA synthesized late in infection included 27S, 24S, 19S, and 11S size classes, all of which annealed to A fragment DNA. Additional RNA migrating as 24S hybridized to E and C fragment DNA, and 23S RNA annealed to F fragment DNA. The results of the hybridizations of size fractionated RNAs with viral DNA fragments enabled formation of a partial map of viral mRNAs with respect to the adenovirus 2 genome. Some of the viral RNAs may represent transcripts which overlap R1 cleavage sites, because in at least three instances hybridization revealed viral RNAs which have the same electrophoretic mobility and anneal to fragments that are contiguous on the genome.

Adenoviridae↗
PubMed1974

Elongation of the polyadenylate segment of messenger RNA in the cytoplasm of mammalian cells.

Chinese hamster and mouse sarcoma 180 ascites cells, treated with high levels of actinomycin D, still carry out limited poly(A) synthesis. The residual activity, which consists of poly(A) chain extension in the cytoplasm as well as in the nucleus, is essentially insensitive to cordycepin. Nuclear polyadenylation proceeds linearly and involves the gradual extension of unusually long poly(A) sequences. Cytoplasmic poly(A) labeling is initially more rapid than in the nucleus, but levels off within 5-10 min. It consists of addition of seven to eight AMP residues to the poly(A) sequences in preexisting mRNA molecules. The levelling off can be accounted for by a rapid turnover of the extremity of the poly(A) chain in the mRNA. Cytoplasmic poly(A) chain extension can be detected in cells not subjected to the actinomycin treatment. The rate of this process is of the same order of magnitude as that of new poly(A) transfer from nucleus to cytoplasm. It could serve to control the length of the poly(A) sequence in mRNA.

Adenine Nucleotides↗
PubMed1974

Formation of Sindbis virus capsid protein in mammalian cell-free extracts programmed with viral messenger RNA.

Extracts from Krebs II ascites cells and rabbit reticulocytes effectively synthesize viral proteins with Sindbis viral mRNA isolated from Sindbis-infected BHK cells. The major product is identical to Sindbis capsid protein on the basis of its electrophoretic mobility in sodium dodecyl sulfate-acrylamide gels and two-dimensional tryptic-peptide fingerprints. Various amounts of several additional discrete polypeptides are formed, depending on the components of the cell-free extracts. One of these polypeptides may be a prematurely terminated part of the viral-capsid protein, while another is larger in molecular weight than capsid protein but contains the capsid tryptic peptides. Several of the proteins formed in vitro also are detected in extracts of Sindbis-infected BHK cells labeled with [(35)S]methionine. The three proteins found in Sindbis virions are postulated to originate by proteolytic cleavage from a larger molecular weight polypeptide precursor that is translated from a polycistronic mRNA presumed to contain a single site for initiation of protein synthesis. The two in vitro systems appear to translate this polycistronic viral mRNA to yield specific viral capsid although no evidence was found for post-translational proteolysis. Other mechanisms for production of the capsid protein in the cell-free extracts are considered, and some of these may function in the viral-infected cell where unusually large amounts of viral capsid proteins are frequently detected.

Animals↗
PubMed1974

Isolation of DNA Strand-specific early messenger RNA species in cells infected by human adenovirus 2.

Hybridization to the separated light (L) and heavy (H) strands of adenovirus 2 DNA in 50% formamide at 37 degrees was used to isolate undegraded virus-specific RNA molecules from the polyribosomes of cycloheximide-treated human KB cells early after infection with adenovirus 2. About 20% of polyribosomal RNA labeled with [(3)H]uridine from 4 to 7 hr after infection was virus-specific. Twice as much labeled RNA was homologous to the L strand as to the H strand. Polyacrylamide gel electrophoresis of RNA selected with unfractionated adenovirus DNA resolved a major component of virus-specific RNA in the 19-20 S region of the gel and smaller amounts of viral RNA in two heterogeneous fractions migrating at 15-18 S and 21-26 S. Selection with individual DNA strands showed that the 19-20 S main size class of early mRNA consists of two homogeneous RNA species with slightly different mobilities, the transcripts from the L and H strand having molecular weights of 7.4 x 10(5) and 7.7 x 10(5), respectively. The 15-18 S RNA hybridized with the L strand and the 21-26 S RNA with the H strand.

Adenoviridae↗
PubMed1974

Methylation of newly synthesized viral messenger RNA by an enzyme in vaccinia virus.

Purified vaccinia virions contain an enzyme that incorporates methyl groups from S-adenosylmethionine into viral RNA synthesized by the core-associated DNA-dependent RNA polymerase. This incorporation, by partially disrupted virions, was dependent on the presence of all four ribonucleoside triphosphates and Mg(++) and was inhibited by actinomycin D. At saturation, 2.3 methyl groups were incorporated per 1000 nucleotides. The methyl-labeled RNA product was sensitive to alkali and ribonucleases and hybridized to filters containing immobilized poly(U) or vaccinia DNA. The methyl groups were not located on the 3'-terminal polyadenylate sequence, nor were they randomly distributed along the RNA chain. The lability of a large portion of the methyl groups to perchloric acid digestion was consistent with an O-methyl linkage, and the chromatographic properties of the alkali-digested material suggested that either the 5'-terminus or up to three consecutive internal nucleotides were methylated. Methylation probably occurs at the macromolecular level, since added vaccinia RNA was a suitable substrate. The failure of heterologous rRNA and tRNA species as well as homopolyribonucleotides to act as substrate suggested that a specific sequence might be required.

Carbon Radioisotopes↗
PubMed1975

Cell-free translation of immunoglobulin messenger RNA from MOPC-315 plasmacytoma and MOPC-315 NR, a variant synthesizing only light chain.

Total poly(A)-containing mRNA was isolated from the MOPC-315 and MOPC-315 NR plasmacytomas. The RNA was further fractionated on sodium dodecyl sulfate-sucrose gradients. The MOPC-315 mRNA fractions directed the synthesis of both the heavy chain and light chain precursor of the MOPC-315 IgA protein in a cell-free extract of Ehrlich ascites tumor cells. None of the MOPC-315 NR mRNA fractions tested programmed the synthesis of the heavy chain in this system. Analysis of cell-free products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoprecipitation demonstrated that no translatable heavy chain mRNA could be extracted from the MOPC-315 NR variant plasmacytoma.

Animals↗
PubMed1975

Methylated nucleotides block 5'-terminus of vaccinia virus messenger RNA.

Studies on the nature and location of the methylated nucleotides in mRNA synthesized in vitro by vaccinia virus particles revealed an unusual 5'-terminal structure. Evidence that the pyrophosphate group is blocked by 7-methylguanosine and that both 2'-O-methyl-adenosine and 2'-O-methylguanosine occupy penultimate positions was presented. According to this model, the 5'-termini of vaccinia virus mRNAs are: 7MeG-5'ppp-5'GMepNp and 7MeG-5'AMepNp.

Adenosine↗
PubMed1975

Short-lived messenger RNA in HeLa cells and its impace on the kinetics of accumulation of cytoplasmic polyadenylate.

Accumulation of [3H]adenine in the acid-soluble pool and in nuclear and cytoplasmic poly(A) of HeLa cells shows that the nuclear poly(A) rises along a curve similar to that of the acid-soluble pool. By use of a [3H]guanosine pulse-chase experiment in adenine-grown cells, at least 35-50% of the pulse-labeled mRNA was found to have a half-life of about 1-2 hr. A mathematical model involving nuclear poly(A) synthesis and conservative transport to the cytoplasm has been derived from the new information about mRNA with a short half-life. This model predicts curves similar to those found for nuclear and cytoplasmic accumulation of poly(A). Thus, there is no necessity on kinetic grounds to invoke either nuclear turnover or cytoplasmic synthesis of poly(A).

Adenine↗
PubMed1975

Coupled in vitro transcription and translation of vesicular stomatitis virus messenger RNA.

The virion transcriptase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) of vesicular stomatitis virus was fully active when ribonucleoprotein cores from purified virions were added to cell-free protein synthesizing systems of eukaryotic origin. Synthesis of mRNA was linear for at least 3 hr and the newly synthesized viral mRNA was efficiently utilized for the synthesis of viral proteins N (nucleoprotein), NS, and M (matrix); small amounts of a putative G (glycoprotein protein precursor and several unidentified polypeptides were regularly synthesized. The ratio of the various newly synthesized viral proteins was identical after different periods of coupled mRNA and protein synthesis. Identical proteins were obtained when the cell-free protein synthesizing systems were programmed with purified VSV mRNA synthesized in vitro. No detectable L protein was synthesized, even though transcripts complementary to the complete viral genome were detectable in the mRNA preparation by hybridization.

Animals↗
PubMed1975

Methylation of messenger RNA of Newcastle disease virus in vitro by a virion-associated enzyme.

Purified Newcastle disease virus contains an enzyme that incorporates the methyl group from S-adenosyl-L-methionine into RNA synthesized in vitro by the virion-associated RNA polymerase (RNA nucleotidyltransferase). Incorporation of radioactivity from S-adenosyl-L-[methyl-3H]methionine was totally dependent upon RNA synthesis. The methylation reaction was completely inhibited by S-adenosyl-L-homocysteine, suggesting the transfer of only the methyl group of S-adenosyl-methionine to RNA products. Velocity sedimentation and hybridization of the in vitro product RNA indicated that both [3H]methyl and [32P]GMP labels resided in single-stranded 18S RNA molecules which were virus specific. Approximately 1 to 2 methyl groups were incorporated per RNA molecule. DEAE-cellulose chromatography of product RNA after alkaline hydrolysis suggested that the 5' terminus was the site of methylation.

Chromatography, DEAE-Cellulose↗
PubMed1976

Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA.

Murine leukemia virus particles assembled in actinomycin D-treated cells were detected by determination of reverse transcriptase [RNA-dependent DNA polymerase (nucleotidyltransferase)] activity and by radioimmunoassay of the major virion protein, p30. The levels of enzyme activity and p30 protein were both 30-40% relative to the control over an 8 hr period, whereas after 3 or 4 hr infectivity was reduced by 95%. Thus, virions produced in the absence of RNA synthesis represent a fairly homogeneous population of defective particles. Although RNA synthesis is not necessary for virus assembly, protein synthesis is required. Treatment of cells with 10 mug/ml of cycloheximide reduced virus production by 80-85% within 2 hr, and by greater than 95% at later times. As might be expected from this finding, viral protein synthesis accompanies virus assembly in actinomycin D-treated cells. Newly synthesized proteins associated with the defective particles were identical with those found in standard virions and were present in the correct proportions. The results demonstrate that viral mRNA persists in cells in which RNA synthesis is blocked and continues to direct viral protein synthesis with a functional half-life of approximately 6-8 hr. Since viral mRNA is not packaged in virions even when viral RNA synthesis is shut off [Levin et al. (1974) J. Virol. 14, 152-161], we propose that murine leukemia virus-infected cells contain two nonequilibrating pools of intracellular viral RNA molecules, one associated with polyribosomes and one which is encapsidated into extracellular particles.

AKR murine leukemia virus↗
PubMed1976

A major species of mammalian messenger RNA lacking a polyadenylate segment.

Translation of total polysomal RNA from sarcoma 180 ascites cells in a wheat germ cell-free system produces two major polypeptides, A and B, with molecular weights of 50,000 and 45,000, respectively. Fractionation on Millipore filters or on oligo(dT)-cellulose leads to retention of the mRNA specific for protein A in the poly(A)-containing fraction and to accumulation of the B mRNA in the unadsorbed poly(A)-deficient fraction. The mRNA for B sediments at approximately 18 S; it is released as a 50S ribonucleorprotein upon EDTA treatment of polysomes. Its translation is particularly sensitive to an inhibitor present in the polysomal RNA. The poly(A)-deficient mRNA for the 45,000 dalton polypeptide is also present in mouse myeloma MPC-11 cells, where it seems to be localized in membrane-bound polysomes.

Animals↗
PubMed1977

Cell-free translation of simian virus 40 early messenger RNA coding for viral T-antigen.

Simian virus 40 (SV40) mRNA was isolated by hybridization of cytoplasmic RNA, from SV40-infected BS-C-1 monkey cells early in lytic infection, to SV40 DNA immobilized on Sepharose. The early viral mRNA, when added to a wheat-germ translation system, directed the synthesis of a unique class of products including a 90,000 molecular weight (Mr) polypeptide. It was found that this 90,000 Mr product as well as a prominent 17,000 Mr polypeptide could be specifically immunoprecipitated with hamster antiserum to SV40 T-antigen, but not with hamster control serum. Similar immunoprecipitation of extracts of SV40-infected cells with hamster anti-T serum yielded 90,000 Mr and 17,000 Mr polypeptides; these polypeptides were not found in immunoprecipitates of uninfected cell extracts. SV40 cRNA, prepared by asymmetric transcription of plaque-purified SV40 DNA, directed the cell-free synthesis of several products, including a 70,000 Mr polypeptide that could be specifically immunoprecipitated with anti-T serum. However, no T-antigen-related polypeptide was found in infected cells that corresponded in size to the major immunoprecipitated cRNA product.

Animals↗
PubMed1979

Segments of simian virus 40 DNA spanning most of the leader sequence of the major late viral messenger RNA are dispensable.

A highly specific procedure for the isolation of deletion mutants is described. The size and location of the deletions can be predetermined. By this method a series of deletion mutants mapping within and near the untranslated 5' leader sequence of the late 16S mRNA of simian virus 40 have been isolated. The boundaries of the deletions have been accurately determined by DNA sequence analysis. The deletions range from 20 to 223 nucleotides. All these deletions mutants are viable and grow without helper virus. The largest of these deletions removes the entire leader sequence except for six nucleotides at the 3' end that are probably involved in covalent linkage with the 5' end of the body of the mRNA located 937 nucleotides away on the genome. Three of the deletion mutants remove the 5' end of the leader that normally bears the cap structure of the mRNA. A large segment immediately preceding the leader sequence is also removed in one of these mutants, ruling out the generation of the 5' end of the mRNA via initiation of transcription at this point. The circularization of linear infecting DNA producing the DNA of the deletion mutants proceeds mainly by way of blunt end ligation in vivo.

Base Sequence↗
PubMed1980

Unusual features in the nucleotide sequence of a cDNA clone derived from the common region of avian sarcoma virus messenger RNA.

We have constructed a recombinant plasmid containing a 700-base pair (bp) cDNA copy of the common region present at the 3' end of Schmidt-Ruppin avian sarcoma virus (ASV) 21S mRNA. The cDNA was inserted into plasmid pBR322 at the Pst I site by the G-C tailing method. A restriction map of the cloned insert from a recombinant plasmid pSRI indicates that it corresponds to the 3' end of the ASV genome. R-loop analysis with ASV genomic RNA indicates that the insert is colinear with the ASV genome over most of its length. The sequence of 331 bp at the 3' end of the DNA insert was determined and shows that the insert contains extra sequences not found at the 3' end of ASV genomic RNA. Following the terminally redundant sequence of 20 bp that has been found at the extreme 3' end of genomic RNA is a sequence of 79 bp that is almost identical to that located immediately next to the 20-bp repeat at the 5' end of ASV genomic RNA. This is followed by 18 bp of unique sequence, possibly of host origin. The structure of the clone suggests that ASV mRNA may differ from genomic RNA at its 3' end and that 21S mRNA is transcribed from integrated ASV DNA and contains at its 3' end sequences derived both from the 5' end of the ASV genome and from host DNA adjacent to the site of integration. The presence of termination codons in all three reading frames suggests that the common region probably does not contain coding sequences. However, the presence of sequences that resemble probable promoter sites supports the possibility that this region may be involved in the regulation of transcription.

Alpharetrovirus↗
PubMed1984

In vitro-synthesized adenovirus 2 messenger RNA precursors are accurately spliced by nuclear extracts.

Precursor mRNAs were synthesized in vitro from a plasmid in which the early region 2 gene of adenovirus 2 is fused to an efficient bacteriophage promoter (Salmonella phage 6). The RNAs were purified and used as substrates for in vitro splicing in the presence of nuclear extracts prepared from MOPC-315 mouse myeloma cells. The in vitro splicing was accurate at the nucleotide level. The reaction occurs rapidly and without any detectable lag. The concentration of the pre-mRNA precursor during incubation appears to be an important factor for high efficiency (60%-80%) of in vitro RNA splicing. Fractionation of the splicing components as well as modifications of the DNA template to study the nucleotide-sequence requirement for in vitro splicing can now be accomplished with this system.

Adenoviruses, Human↗