13S mRNA Research Articles

A novel adenovirus-2 E1A mRNA encoding a protein with transcription activation properties.

Two novel adenovirus-2 early region 1A mRNAs, designated 10S and 11S, have been characterized. They differ from the previously described 9S, 12S and 13S mRNAs by having an additional intron removed during mRNA maturation. The 10S and 11S mRNAs encode proteins with mol. wts of 30 and 35 kd. These proteins are encoded in the same translational reading frame as the 12S and 13S mRNA products and differ by lacking 72 amino acids between position 27 and 98. A functional analysis showed that both the 10S and 11S mRNA products are non-essential for lytic virus growth, and, furthermore, defective in cellular transformation. Interestingly the 11S mRNA product functioned as an efficient transcriptional activator in transient expression assays but was very ineffective as a gene activator during virus growth. Moreover, the virus expressing the 11S cDNA failed to block host cell gene expression although substantial amounts of late proteins were expressed. From the biological properties of the E1A cDNA mutants it was possible to localize two functional domains in the E1A proteins; one region required for transcriptional activation (amino acids 140-185), and a second domain required for adenovirus transformation and the control of viral and cellular gene expression during a lytic infection (amino acids 27-98).

Alternative splicing of E1A transcripts of adenovirus requires appropriate ionic conditions in vitro

We have developed an in vitro splicing system using a HeLa cell nuclear extract that is highly active for the alternative splicing of the natural E1A transcripts. The efficiency of using the three alternative 5′ splice sites is strongly dependent on the ionic conditions in the reaction, and the simultaneous production of the 13S, 12S, and 9S mRNA species is observed only at appropriate salt concentrations. All the intermediate and final splicing products have been extensively characterized and it has been demonstrated that the same major branch site is used for all the alternative reactions. The ratio of 13S to 9S mRNAs formed is close to that observed in vivo early in infection, suggesting that most of the mechanisms giving rise to alternative splicing are preserved in vitro.

Posttranscriptional regulation of ribosomal protein S20 and stability of the S20 mRNA species

I have tested whether selective degradation of mRNA for ribosomal protein S20 of Escherichia coli occurs under conditions for which the expression of S20 is regulated posttranscriptionally. Blot hybridization of total RNA extracted from cultures at different times after addition of rifampin has permitted the estimation of relative levels of the two S20 mRNA species and their half-lives. In a strain harboring a plasmid containing the complete gene for S20, including the transcriptional terminator, moderate posttranscriptional repression of S20 synthesis is accompanied by a substantial increase in the half-lives of both S20 mRNAs relative to those in the haploid parental strain. In an otherwise identical strain in which the transcriptional terminator is deleted from the plasmid-borne S20 genes, the half-life of total S20 mRNA declines more than twofold relative to that in the haploid parent. Thus accelerated decay of the mRNAs for ribosomal protein S20 appears to be an artifact of deletion of the transcriptional terminator, rather than a physiologically significant consequence of translational repression.

Developmentally controlled expression of a gene associated with self-incompatibility in Nicotiana alata

In many flowering plant species, a system of self-incompatibility, typically controlled by a single gene with multiple alleles (S-gene)1, enables individual plants to recognize and reject their own pollen. In gametophytically determined systems, the growth of self-pollen tubes is arrested in the style or occasionally the ovary. The expression of this self-incompatibility is developmentally regulated, being strongest in mature flowers and weak in immature styles1,2. We have used a complementary DNA clone, believed to encode the S2-allele of Nicotiana alata2,3, to detect S2 messenger RNA by in situ hybridization to sections. We report here that expression of the gene occurs in the stigma and throughout the secretory tissue, but not in other parts, of mature pistils. In immature flowers, S2 mRNA is confined to the proliferated epidermis of the stigma. We conclude that S2-gene expression correlates well with the expression of incompatibility.

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells.

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.

Simian virus 40 agnoprotein facilitates normal nuclear location of the major capsid polypeptide and cell-to-cell spread of virus

The simian virus 40 agnoprotein is a 61-amino-acid, highly basic polypeptide that is coded within the 5' leader of late 16S mRNAs. To better understand agnoprotein function and to more effectively differentiate cis-from trans-acting effects of an agnogene mutation, we constructed a mutant virus that carries a single-base-pair substitution and fails to produce agnoprotein. pm 1493 contains a T/A to A/T transversion at sequence position 335. This mutation converts the agnoprotein initiation codon from ATG to TTG, preventing synthesis of the protein. The mutant displays only a modest growth defect in CV-1P and AGMK cells and no defect in BSC-1 cells. Early-gene expression, DNA replication, synthesis of late viral products, and the kinetics of virion assembly all appear normal in pm 1493-infected CV-1P cells. Immunofluorescent studies, however, indicate that localization of the major capsid polypeptide VP1 is different in mutant- than wild-type virus-infected cells. Furthermore, the lack of agnoprotein led to inefficient release of mature virus from the infected cell. Agnogene mutants could be severely compromised in their ability to propagate in monkeys given their reduced capacity for cell-to-cell spread.

Biosynthesis of reovirus-specified polypeptides: Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain bicistronic s1 mRNA which encodes the minor capsid polypeptide σ1a and the nonstructural polypeptide σ1bNS

Human reovirus serotype 1 Lang strain s1 mRNA, which encodes the minor capsid cell attachment protein sigma 1a and the nonstructural protein sigma 1bNS, was cloned as a cDNA:mRNA heteroduplex in Eschericia coli using phage M13. The Lang strain s1 mRNA is 1462 nucleotides in length and possesses two open reading frames. The first begins at nt 14 and has a coding capacity of 418 amino acids, sufficient to account for sigma 1a; the second begins at nt 75 and has a coding capacity of 119 amino acids, sufficient to account for sigma 1bNS. Comparison of the Lang serotype s1 sequence derived from cDNA clones of s1 mRNA with the Lang S1 sequence derived from cDNA clones of the S1 dsRNA genome segment definitively establishes that reovirus plus-strand mRNA is structurally equivalent to the plus-strand of the dsRNA genome segment.

A low-molecular-weight RNA from mouse ascites cells that hybridizes to both 18S rRNA and mRNA sequences.

A low-molecular-weight RNA species from mouse ascites cells has been selected and purified by its intermolecular RNA X RNA hybridization capabilities. This 4.5S RNA is able to base pair with poly(A)+ mRNA sequences and with 18S rRNA. Melting experiments have shown that the intermolecular hybrids formed with this complementary low-molecular-weight RNA are of comparable stability to other RNA X RNA interactions. Analysis has shown that this hybridizing RNA is 87 nucleotides long and has an unusual sequence structure. Located near the 3' terminus is an alternating pyrimidine dinucleotide region of UUCCUUCCUU. This region along with the 3'-adjacent nucleotides form a 14-nucleotide sequence that exhibits perfect complementarity with 18S rRNA. An additional region of 10 nucleotides at the 3' terminus is perfectly homologous to a similarly located sequence in 5.8S rRNA. An obvious RNA polymerase III binding site is not found internally in this low-molecular-weight RNA sequence. The complementary and homologous character of hybridizing RNA with respect to rRNA and mRNA sequences suggests a potential regulatory role for this RNA in the coupling of ribosome and mRNA functions.

Molecular determinants of alphavirus neurovirulence: nucleotide and deduced protein sequence changes during attenuation of Venezuelan equine encephalitis virus.

The nucleotide and deduced amino acid sequences of the structural proteins of the TC-83 vaccine strain of Venezuelan equine encephalitis (VEE) virus have been determined from a cDNA clone containing the 26S mRNA coding region. A cDNA clone encoding the equivalent region of the virulent parent VEE virus [Trinidad donkey strain (TRD)] has been sequenced previously. Comparison of the sequences of the TC-83 and TRD cDNA clones revealed 13 nucleotide differences. Neither the organization of the structural proteins (5'-capsid-E3-E2-6K-E1-3') nor the length (3762 nucleotides) of the open reading frame coding for the viral polyprotein precursor was altered during attenuation. Of the 13 nucleotide differences between the cDNA clones of TC-83 and TRD, nine occurred in the dominant population of the respective genomic RNAs from plaque-purified viruses. Six of the nine mutations were clustered in the E2 surface glycoprotein gene. All five of the nucleotide changes which produced non-conservative amino acid substitutions in the encoded proteins were located in the E2 gene. Two mutations occurred in the E1 glycoprotein gene; one was silent and the other did not alter the chemical character of the E1 protein. One nucleotide difference was found in the non-coding region immediately preceding the 5'-end of the 26S mRNA. The E2 and non-coding region mutations are candidates for the molecular determinants of VEE virus neurovirulence.

Adenovirus type 12 E1A protein expressed in Escherichia coli is functional upon transfer by microinjection or protoplast fusion into mammalian cells.

We efficiently expressed, in Escherichia coli, and purified the protein product encoded by the human adenovirus type 12 (Ad12) 13S mRNA. The functional properties of the E1A protein were analyzed by introducing the protein by microinjection or protoplast fusion into living mammalian cells. We showed that the E. coli-expressed E1A protein induces gene expression of the adenovirus type 5 (Ad5) E1A deletion mutant Ad5dl312. The purified E1A protein rapidly and quantitatively localized to the cell nucleus after microinjection into the cytoplasm. In addition, we raised high-titered monospecific antibodies to the purified Ad12 E1A protein. Using deleted forms of an adenovirus type 2 and Ad5 hybrid (Ad2/5) E1A protein, we showed that all of the epitopes conserved between Ad2/5 E1A and Ad12 E1A protein that are recognized by the Ad12 E1A-specific antiserum map to within the first exon-encoded amino-terminal half of the protein.

Adenovirus-2 early region IA protein synthesized in Escherichia coli extracts indirectly associates with DNA.

The interaction of adenovirus-2 (Ad2) early region IA (EIA) protein (encoded by the 13S mRNA) with DNA was examined using EIA protein synthesized in Escherichia coli extracts directed by a plasmid containing the cloned EIA gene. Without any purification, this protein when chromatographed over calf thymus DNA immobilized on cellulose, showed at least two types of salt-sensitive activities after associating with equal efficiency to both single- and double-stranded DNA; however, a putative C-terminal proteolytic fragment of the EIA protein (identified by immunoprecipitation with anti-serum specific to the EIA carboxy-terminus) showed 10-fold greater affinity to double- versus single-stranded DNA. When examined with Ad2 DNA, the EIA protein had a retention that was at least 2-fold higher compared to calf thymus DNA, suggesting some substrate specificity. It was also found that a 1.0 M salt concentration was required for the elution of the EIA protein from pBR322 DNA containing cloned regulatory sequences of adenovirus early regions II and III. This suggests that the strength of the protein interaction depends on the target DNA sequence. Finally, addition of uninfected HeLa cell extract to bacterial extracts containing EIA-like protein potentiated the association of the protein to double-stranded calf thymus DNA up to 7-fold. These data support the hypothesis that the EIA protein interacts with target DNA, presumably mediated by co-factor(s) in an indirect fashion.

Additional methylation at the N(2)-position of the cap of 26S Semliki Forest virus late mRNA and initiation of translation

Cap analysis of the late 26S Semliki Forest viral mRNA reveals that almost 30% of the caps possess both a methyl group at the N(7)-position and one at the N(2)-position. We have compared the degree of methylation of the caps of polysomal and non-polysomal 26S mRNA in order to check whether this feature is responsible for its translation late in infection. It was found that extra methyl groups on the caps cause a lower rate of initiation. Polysomal 26S mRNA contained less m 2,7G- and m 2,2,7G-caps than free 26S. The cap analog m 2,2,7Gp was slightly less inhibitory than m 7Gp in an in vitro translation system.

Modulation of the level of expression of cellular genes in adenovirus 12-infected and transformed human cells.

The level of expression of thymidine kinase (TK), heat shock protein 70 (HSP70), beta-tubulin and p53 was assessed in human embryo kidney cells (HEKs) infected with adenovirus type 12 (Ad 12) and Ad 12 early region 1 (E1) mutants. HSP70, beta-tubulin and p53 levels were unchanged but TK activity was dramatically increased following wild-type infection. The initial activation of TK required the expression of the product of the E1A 13S mRNA but sustained expression only occurred with those viruses expressing the E1B proteins as well. A number of human cell lines transformed with either Ad 12 or Ad 5 E1 DNA were also assessed for the level of expression of HSP70, beta-tubulin and p53. Both HSP70 and beta-tubulin levels were greatly increased compared with primary human cells although there was considerable variation between lines. p53 was only expressed at high levels in Ad 12-transformed lines expressing E1A and E1B proteins.

Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen.

The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K). All three forms were phosphorylated and were present in both the nucleus and the cytoplasm. The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with [35S]methionine in Ad12-infected cells. The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen. Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens. Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus. Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the DNA sequence, whereas no methionine was released from the 39K T antigen during the first six cycles of Edman degradation. We propose that the short-lived 43K T antigen is the primary product of the 13S mRNA, the 266R T antigen; the somewhat more stable 42K T antigen is the primary product of the 12S mRNA, the 235R T antigen.(ABSTRACT TRUNCATED AT 400 WORDS)

Biosynthesis of reovirus-specified polypeptides: Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s4 mRNA which encodes the major capsid surface polypeptide σ3

Serotype 1 Lang strain s4 mRNA, which encodes the major capsid surface polypeptide σ3 of reovirions, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage Ml3. A complete consensus nucleotide sequence for s4 mRNA has been determined from cDNA clones. The Lang strain s4 mRNA is 1196 nucleotides in length and possesses an open reading frame with a coding capacity of 365 amino acids, sufficient to account for a σ3 polypeptide of 41,212 daltons. Comparison of the serotype 1 (Lang) s4 sequence with the serotype 3 (Dearing) S4 sequence reveals 94% homology at the nucleotide level; the predicted σ3 polypeptides of the Lang and Dearing strains display 96% homology at the amino acid level. Two third base C codons (leu:CUC and ser:AGC) are used about one-tenth as frequently in the reovirus s4 mRNAs as compared to mammalian cellular mRNAs.

Lytic and transforming functions of individual products of the adenovirus E1A gene.

To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA E1A region but not in the presence of a 12S E1A cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the E1A 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.

Molecular cloning and sequence analysis of a haploid expressed gene encoding t complex polypeptide 1

The mouse t haplotypes show defects in spermatogenesis attributed to multiple loci on chromosome 17. We have cloned the gene for an abundant testicular germ cell protein, t complex polypeptide 1, which has a variant form in t haplotypes, TCP-1A. A cDNA clone, pB1.4, which hybridizes to a 19S mRNA that is abundant in haploid cells during mouse spermatogenesis, derives from the 3′ end of the mRNA encoding TCP-1B. The Tcp-1 gene appears to be a member of a novel gene family and shows multiple changes between the predicted amino acid sequences of TCP-1B and TCP-1A. An additional Taq1 site is created by a T to C transition in the predicted open reading frame of the Tcp-1 a gene. The resultant RFLP has allowed typing of the Tcp-1 gene cluster in 54 complete and partial t haplotype chromosomes. DNA sequence comparison of the Tcp-1 genes suggests that the t haplotype chromosome arose within the genus Mus more than one million years ago.

An insertion mutation in the adenovirus type 12 early region 1A 13S mRNA unique region.

An adenovirus type 12 mutant, in203S, was constructed to contain an insertion of two amino acids in the early region 1A (E1A) 13S mRNA-coding region and in the E1A 12S mRNA intron. in203S could not grow in HeLa and KB cells. Virus DNA replication was scarcely detected at a low multiplicity of infection, but was detected at a high multiplicity of infection. The transcription of early genes other than E1A was not detected at 13 h after infection, but became detectable after longer incubation. The transcription of the E1A gene was also reduced to about one-fifth of the wild-type level. The mutant induced fewer foci of smaller sizes than the wild type in rat 3Y1 and secondary rat kidney cells. The induction of cellular DNA synthesis was reduced in rat 3Y1 cells infected with in203S as compared with that in wild type-infected cells. These results show that the E1A 13S mRNA-derived polypeptide of adenovirus type 12 is required for activation of early genes, cell transformation, and induction of cellular DNA synthesis.

The storage of globin mRNA during the inhibition of protein synthesis by heme deprivation

The inhibition of protein synthesis in reticulocytes and their lysates caused by heme-deprivation is reversible on restoration of an optimal heme concentration. Inhibition is accompanied by the disaggregation of polyribosomes and the accumulation of components of the translational mechanism. By determining the fate of labeled globin 9S mRNA added to an unfractionated reticulocyte lysate cell-free system, we find that normal cellular mRNA accumulates during inhibition in 20S and 48S complexes and in a complex which sediments just ahead of the 80S ribosome dimer OD 260 peak (designated as ≥80S complex) 1 1 The “S-values” given in this paper are approximate values based on the sedimentation of the designated complex in the isokinetic sucrose gradients used in this study. The markers used for calibration were 9S globin mRNA, the 40S and 60S ribosomal subunits and the 80S initiation complex. . The 20S and ≥ 80S complexes are the major pools of stored mRNA which is readily translated if optimal heme conditions are restored. In the 48S complex, however, the mRNA remains non-functional, and the complex is abortive, probably as a result of deacylation of the Met. tRNA f.

Sequence-specific activation of transcription by adenovirus EIa products is observed in HeLa cells but not in 293 cells.

The adenovirus EIa gene products activate transcription from the viral EIII and EIIaE promoters. We studied the mechanism of this stimulation by constructing a series of chimeric promoter recombinants containing the upstream regions of the EIII and EIIaE promoters linked to the TATA box-start-site regions of the viral major late and EIIa late promoters. By introducing these recombinants into HeLa cells together with recombinants producing the EIa gene products, we demonstrated that the induction of EIII and EIIaE transcription by EIa 13S and 12S mRNA products is dependent on sequences located in the upstream region (approximately -40 to -250) of these promoters. In addition, we showed that the major late and EIIa late upstream promoter regions do not contain such EIa-responsive sequence elements. In contrast, after transfection of these chimeric promoter recombinants into 293 cells (which constitutively express the EIa proteins), we found that their relative levels of transcription are similar and markedly different from those observed when they are cotransfected into HeLa cells with EIa protein-producing recombinants. We conclude that the efficiency of transcription from a given promoter in 293 cells is not necessarily related to the presence of a specific EIa-responsive element.