13S mRNA Research Articles

Role of signal recognition particle in the membrane assembly of Sindbis viral glycoproteins.

We have investigated the role of signal recognition particle (SRP) in the biosynthesis of Sindbis glycoproteins by translating the viral 26S mRNA in a wheat-germ cell-free system. SRP was shown to have no effect on the synthesis or proteolytic processing of the cytoplasmic C protein. In contrast, the membrane integration and the proteolytic processing of the viral glycoproteins PE2 and E1 were demonstrated to be SRP-dependent. In the absence of microsomal membranes, SRP caused an arrest of the synthesis of the viral glycoproteins. This arrest could be released by the addition of salt-extracted microsomal membranes. Synchronization experiments indicated that the uncleaved signal sequence of PE2 was recognized by SRP after at most 130 amino acids of PE2 had been polymerized. No apparent interaction of SRP with a putative signal sequence of E1 and/or a 6-kDa peptide could be detected.

Peptide maps and N-terminal sequences of polypeptides from early region 1A of human adenovirus 5.

Experiments exploring the reasons for a multiplicity of products from early region 1A of adenovirus 5 are described. Labeled early region 1A products from wild-type virus were synthesized in infected cells and in a cell-free system programmed with mRNA from infected cells, immunoprecipitated specifically with an antipeptide serum, E1A-C1, directed against the C-terminal sequence of E1A products, and separated by gel electrophoresis. Two-dimensional maps of [35S]methionine-labeled peptides were consistent with antigens of 52,000 daltons (52K) and 48.5K being from the 13S mRNA and antigens of 50K, 45K, and 35K from the 12S mRNA. Partial N-terminal sequences of 52K, 50K, 48.5K, and 45K synthesized in vitro showed that each of these antigens was initiated at the predicted ATG at nucleotide 560 in the DNA sequence. These results eliminate multiple initiation sites and proteolytic cleavage at the N-terminal end as sources of antigen diversity. Peptide maps and N-terminal sequences were obtained in a similar way for E1A products from the Ad5 deletion mutant dl1504, which lacks the normal initiator codon. As predicted, these polypeptides are initiated at the next ATG, 15 codons downstream in the wild-type sequence. These results are discussed in relation to Kozak's ribosomal scanning model.

The number of ribosomes on simian virus 40 late 16S mRNA is determined in part by the nucleotide sequence of its leader.

The size distributions of polyribosomes containing each of three simian virus 40 late 16S mRNA species that differ in nucleotide sequence only within their leaders were determined. The two 16S RNA species with shorter leaders were incorporated into polysomes that were both larger (on average) and narrower in size distribution than was the predominant wild-type 16S RNA. Therefore, the nucleotide sequence of the leader can influence the number of ribosomes present on the body of an mRNA molecule. We propose a model in which the excision from leaders of sizeable translatable regions permits more frequent utilization of internally located translation initiation signals, thereby enabling genes encoded within the bodies of polygenic mRNAs to be translated at higher rates. In addition, the data provide the first direct evidence that VP1 can, indeed, be synthesized in vivo from the species of 16S mRNA that also encodes the 61-amino acid leader protein.

The number of ribosomes on simian virus 40 late 16S mRNA is determined in part by the nucleotide sequence of its leader.

The size distributions of polyribosomes containing each of three simian virus 40 late 16S mRNA species that differ in nucleotide sequence only within their leaders were determined. The two 16S RNA species with shorter leaders were incorporated into polysomes that were both larger (on average) and narrower in size distribution than was the predominant wild-type 16S RNA. Therefore, the nucleotide sequence of the leader can influence the number of ribosomes present on the body of an mRNA molecule. We propose a model in which the excision from leaders of sizeable translatable regions permits more frequent utilization of internally located translation initiation signals, thereby enabling genes encoded within the bodies of polygenic mRNAs to be translated at higher rates. In addition, the data provide the first direct evidence that VP1 can, indeed, be synthesized in vivo from the species of 16S mRNA that also encodes the 61-amino acid leader protein.

Adenovirus 2 early region 1A stimulates expression of both viral and cellular genes.

The ability of products from the adenovirus early region 1A to stimulate viral and cellular gene expression has been studied, using a transient expression assay in HeLa cells. We show that the E1A 13S mRNA encodes a diffusible product which is capable of stimulating transcription of adenovirus genes as well as the rabbit beta-globin gene. The E1A 12S mRNA has no detectable stimulatory effect on either cellular or viral genes. Although being able to stimulate both types of genes, we find that the E1A regulatory protein enhances viral gene expression approximately 10 times more than beta-globin gene expression. We also find that when connected to the cis-acting SV40 enhancer element, the beta-globin gene cannot be further stimulated by the trans-acting E1A product. Finally, we find that transfection of either adenovirus or the beta-globin gene into 293 cells, which constitutively expresses the E1A gene products, leads to an enhanced expression which is 10- to 20-fold higher than obtained by co-transfection of HeLa cells. The 293 cells thus provide a simple assay to demonstrate E1A-mediated transcriptional regulation.

Functional roles of two polypeptide chains that compose an antigen-specific suppressor T cell factor.

The functional roles of the two polypeptide chains that compose the T cell suppressor factor (TsF) that mediates the antigen-specific and genetically restricted suppressor function were studied by using the heavy or light chains isolated from the conventional TsF or the 11S and 13S mRNA translation products of TsF. Either the heavy or the light chain of mRNA translation products reconstitutes the active TsF that suppresses the antibody response in an antigen-specific and genetically restricted manner when it is combined with the isolated heavy or light chain from the conventional TsF. As a consequence, the antigen-binding heavy chain mediates the antigen specificity of TsF. On the other hand, the I-J-positive light chain works as an element to determine the genetic restriction specificity. Thus, the identity of the histocompatibility between the I-J haplotypes on the light chain and the responding cell is essential for the functional expression of TsF. No genetic preference, however, was observed, in the association of the heavy and light chains of TsF.

Common and distinct regions of defective-interfering RNAs of Sindbis virus.

Defective-interfering (DI) particles are helper-dependent deletion mutants which interfere specifically with the replication of the homologous standard virus. Serial passaging of alphaviruses in cultured cells leads to the accumulation of DI particles whose genomic RNAs are heterogeneous in size and sequence composition. In an effort to examine the sequence organization of an individual DI RNA species generated from Sindbis virus, we isolated and sequenced a representative cDNA clone derived from a Sindbis DI RNA population. Our data showed that: (i) the 3' end of the DI RNA template was identical to the 50 nucleotides at the 3' end of the standard RNA; (ii) the majority (75%) of the DI RNA template was derived from the 1,200 5'-terminal nucleotides of the standard RNA and included repeats of these sequences; and (iii) the 5' end of the DI RNA template was not derived from the standard RNA, but is nearly identical to a cellular tRNAAsp (S. S. Monroe and S. Schlesinger, Proc. Natl. Acad. Sci. U.S.A. 80:3279-3283, 1983). We have also utilized restriction fragments from cloned DNAs to probe by blot hybridization for the presence of conserved sequences in several independently derived DI RNA populations. These studies indicated that: (i) a 51-nucleotide conserved sequence located close to the 5' end of several alphavirus RNAs was most likely retained in the DI RNAs; (ii) the junction region containing the 5' end of the subgenomic 26S mRNA was deleted from the DI RNAs; and (iii) the presence of tRNAAsp sequences was a common occurrence in Sindbis virus DI RNAs derived by passaging in chicken embryo fibroblasts.

A transcript from the S segment of the Germiston bunyavirus is uncapped and codes for the nucleoprotein and a nonstructural protein.

Analysis of the RNAs present in BHK-21 cells infected with Germiston virus showed that the transcripts from the L and M segments have a size similar to that of their template, whereas two types of complementary RNA are transcribed from the S segment. One, S1, is a full-length "plus" RNA strand (antigenome), and the other, S2, is an incomplete plus RNA strand which serves as mRNA for at least the synthesis of the N protein and a virus-specific nonstructural polypeptide, p12. The 5' ends of these two transcripts appeared to be identical and complementary to the 3' ends of the viral RNA. Our results suggest that transcription of the S fragment either stops 100 to 150 nucleotides from the 5' end of the template, generating an S2 molecule, or continues, generating an S1 molecule. Neither the S1 antigenome nor the S2 mRNA molecules were polyadenylated at their 3' ends or capped at their 5' ends.

Two initiation sites detected in the small s1 species of reovirus mRNA by dipeptide synthesis in vitro.

Reovirus mRNAs directed the synthesis of fMet dipeptides in a translation initiation system reconstituted from rabbit reticulocyte initiation and elongation factors, Artemia salina 80S ribosomes, yeast fMet-tRNAiMet and Escherichia coli3H-labeled aminoacyl tRNAs. As predicted from the GC(U,G) codon that follows the 5'-proximal AUG in half of the viral mRNA species, fMet-Ala was the predominant dipeptide product obtained in response to a mixture of mRNAs or to the separated size classes of medium (m) and small (s) mRNA. The four individual small mRNA species each directed the synthesis of an fMet dipeptide that was consistent with the utilization of the 5'-proximal AUG for initiation. In addition to fMet-Asp, the s1 mRNA also directed fMet-Glu synthesis indicative of initiation in a second reading frame at the 5'-penultimate AUG. The tripeptide fMet-Glu-Tyr was also synthesized from s1 mRNA, which further verified this second initiation site. mRNAs containing 5'-terminal GpppG were 10-15% as active as the corresponding m7G-capped templates. The dipeptide assay provides a rapid method for determining initiation sites in individual mRNAs or in mixtures of mRNAs.

Infection of neuroblastoma cells by Semliki Forest virus. The interference of viral capsid protein with the binding of host messenger RNAs into initiation complexes is the cause of the shut-off of host protein synthesis.

From ribosomal washes of neuroblastoma cells infected with Semliki Forest virus (SFV) a protein of Mr 33000 was purified, which comigrated with the viral capsid protein on sodium dodecyl sulfate/polyacrylamide gels and was recognized by antibodies against the capsid protein of SFV. This protein selectively inhibits the translation of host and early viral 42S mRNA in vitro, but has no effect on late viral 26S and encephalomyocarditis virus mRNA translation. Eukaryotic initiation factor 4B and cap-binding protein restore the translation of host and 42S mRNA to control levels. The capsid protein specifically prevents the binding of host mRNA into 80S initiation complexes, but has no effect on that of late viral mRNA. We propose that the capsid protein is the component responsible for the shut-off of host protein synthesis in SFV-infected cells and for the decreased translational activity of the crude ribosomal washes from these cells.

Gene order of translation of the flavivirus Kunjin: Further evidence of internal initiation in vivo

The rates of inactivation of synthesis of individual virus-specified proteins by ultraviolet radiation provided an estimate of the target sizes of individual viral genes. In a control experiment with Semliki Forest virus, the genes for the structural proteins mapped in the known sequence 5' C-PE2-E1 3', and in accordance with initiation of translation from a single site on 26-S mRNA. Under the same conditions, the inactivation of synthesis of seven Kunjin virus-specified proteins also followed first-order kinetics, but the largest target size was equivalent to only about half the length of the genome. No gene sequence could be deduced using the premise that translation was initiated at a single site. However, genes for the major non-structural proteins could be mapped in the order 5' P98-P71-P10 3' from a postulated ribosomal attachment site about midway along the RNA. The genes for the structural proteins C and E could then be mapped in a preceding sequence 5'…C-E-GP19-P21 3', with a vacant upstream region sufficient to code for at least one of two flavivirus genes not expressed in these experiments. The implications of the two postulated translational units are discussed in relation to previous data on translation strategy of flaviviruses.

Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli.

Plasmid vectors were constructed which expressed three adenovirus tumor antigens fused to a portion of the trpE protein of Escherichia coli. Insertion of adenovirus type 2 DNA from early region 1A (E1A) into such a plasmid led to a fusion protein which contained the C-terminal 266 amino acids of the 289-amino acid protein encoded by the viral 13S mRNA. Similarly, insertion of adenovirus type 5 DNA corresponding to the E1B 55- and 21-kilodalton proteins led to production of fusion proteins containing amino acid sequences from these proteins. After induction with indoleacrylic acid, fusion proteins accumulated stably in the E. coli cells. By using a simple extraction of insoluble protein, 1 to 10 mg of fusion protein per liter of culture was obtained. The fusion proteins were purified on preparative polyacrylamide gels and used to immunize rabbits. Specific antisera for the E1A 289- and closely related 243-amino acid proteins and the E1B 55- and 21-kilodalton proteins were obtained. These sera were used to immunoprecipitate the tumor antigens in cells infected with wild-type and various mutants of adenovirus or to analyze them by an immunoblotting procedure. Mutant E1A proteins in which the C-terminal 70 amino acids are deleted were phosphorylated to much lower extents than the wild-type E1A proteins. This indicates that the deleted region is important for the process of phosphorylation. The E1A proteins were extracted, sedimented in glycerol gradients, analyzed by immunoprecipitation, and found to sediment primarily as monomers.

Elimination of mRNA splicing by a point mutation outside the conserved GU at 5' splice sites.

Nearly all mRNA introns begin with the dinucleotide GU. Mutations in either of these virtually invariant bases have been found to inactivate the corresponding 5' splice site. Until now single base changes in neighboring bases have not been found to completely inactivate a 5' splice site. Here we show that a single A----U transversion in the third position of the adenovirus 2 E1A 13S mRNA intron does prevent RNA splicing at the corresponding 5' splice site.

Purification of mRNA for immunoglobulin κ-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA

The principle of mRNA purification by hybridization to an immobilized DNA fragment was applied to the isolation of mRNA coding for immunoglobulin κ-chains of mouse myeloma MOPC 21 and mouse hybridoma PTF-02. The DNA fragment comprising the 3′-untranslated region and a part of the constant region of the κ-chain gene was covalently attached to diazobenzyloxymethyl-cellulose and used as an affinity adsorbent. A homogeneous 14S mRNA species was obtained by hybridization of total mRNA to the affinity adsorbent at 52°C and by elution at 60 °C. Addition of the purified mRNA to a fractionated cell-free translation system resulted in a significant increase in the radioactivity immunoprecipitated by pig anti-mouse immunoglobulin antibodies. A single radioactive polypeptide of apparent M r of 25,000, corresponding obviously to the κ-chain, was identified as the only translation product.

Direct evidence for the expression of integrated hepatitis B virus DNA in human hepatoma tissue.

The intracellular structure and expression of integrated hepatitis B virus (HBV) DNA in the hepatoma tissue #1707 obtained from an 11-year-old boy were studied. It was found that most of the HBV genome was present in the #1707 DNA. Virus-specific 21S mRNA was also demonstrated by blot hybridization. This is the first direct evidence for the expression of the integrated viral genome in human hepatoma tissue.

Tumorigenicity of cells transformed by adenovirus type 12 by evasion of T-cell immunity.

Evidence is presented that cells transformed by adenovirus type 12 are oncogenic because they escape from T-cell immunity. This effect is brought about by reducing the expression of class I transplantation antigens and is a function of the protein translated from the 13S mRNA, transcribed from early region 1a. These findings establish a novel mechanism by which transformed cells can acquire an oncogenic phenotype.

Evidence of transcription from the late region of the integrated simian virus 40 genome in transformed cells: location of the 5' ends of late transcripts in cells abortively infected and in cells transformed by simian virus 40.

By means of S1 mapping, we observed that spliced 16S and 19S viral late mRNAs--in addition to early mRNAs--were present in cytoplasmic polyadenylated RNA preparations from simian virus 40-transformed cell lines of rat or mouse origin containing no detectable amount of free viral DNA. The amounts of early and late virus-specific mRNAs in these lines were quantified by hybridization of radioactive cytoplasmic polyadenylated RNA with cloned region-specific restriction fragments. The relative amount of late viral mRNA produced in these transformed cells was found to be of the same order as that produced in simian virus 40-infected, nonpermissive baby mouse kidney cells. Moreover, by using the S1 nuclease protection method, we compared the 5' ends of late mRNAs produced (i) in transformed cells, (ii) in abortively infected mouse cells, and (iii) in the late phase of the lytic cycle. The 5' ends of late mRNAs both in abortively infected and in transformed cells were less heterogeneous than the 5' ends of late mRNAs produced during the lytic cycle; however, they were a subset of the 5' ends of late transcripts produced in the lytic cycle.

Attenuation and modulation of mRNA secondary structure in a feedback control system regulating SV40 gene expression

Alternative secondary structures can be predicted for the initial 94 nucleotides synthesized from the major transcription initiation site of SV40 late RNA: a transcription-termination conformation results in the production of aborted RNA and a readthrough conformation leads to the synthesis of the primary SV40 late RNA. In the cytoplasm similar alternative conformations can be predicted for the initial nucleotides at the 5' ends o both the major 16S and 19S late mRNAs. In one of these alternative conformations the AUG initiation codon of the leader protein (agnoprotein) is sequestered and not available for ribosome binding. In the alternative conformation the same AUG is accessible for ribosome binding. We suggest that these mutually exclusive conformations are fundamental elements in a transcription and translation feedback control mechanism regulating the synthesis of 16S and 19S mRNA in the nucleus and the translation of their encoded proteins in the cytoplasm.

Splicing of adenovirus 2 early region 1A mRNAs is non-sequential

The r-strand of early region 1A (E1A) of adenovirus serotype 2 is transcribed into three completely overlapping messenger RNA species, a 9S, a 12S and a 13S mRNA. These three mRNAs are processed from a common colinear RNA precursor and differ only with regard to the size of the intron removed during mRNA maturation. We have studied the processing pathways for the E1A mRNAs by using an assay for transient expression of recombinant plasmids containing the E1A region. All three region E1A mRNAs are synthesized and transported to the cytoplasm in sufficient quantities to permit a detailed study of their structure by S1 endonuclease analysis and primer extension. Additionally, we show that the 72 base-pair repeat from simian virus 40 (SV40), when located upstream of the E1A promoter stimulates expression of the E1A mRNAs five- to tenfold. In order to determine whether splicing of the E1A mRNAs is sequential, i.e. whether the 13S and 12S mRNAs can serve as intermediates in splicing, we constructed two plasmids that lack the intervening sequences that are removed during the maturation of the 12S and 13S mRNAs, respectively. From an analysis of the RNAs produced after transfection with these deletion mutants, the following major conclusions can be made. (1) Splicing of the E1A mRNAs is non-sequential, i.e. the 13S, 12S and 9S RNAs are generated by separate splicing events using the nuclear colinear transcript as the only precursor RNA. (2) RNA splicing is not a prerequisite for an efficient transport of the E1A mRNAs to the cytoplasm. (3) The 13S RNA can be further processed to 12S and 9S RNA species. These splicing events are, however, illegitimate and give rise to 12S and 9S RNAs that both lack one nucleotide at the splice junction. (4) A coupling between splicing and nuclear transport is most likely required in vivo to prevent illegitimate splicing of the 13S mRNA. (5) The 12S RNA does not serve as a precursor for further processing to the 9S RNA. (6) Splicing of the E1A mRNAs followed strictly the G-T-A-G rule.

Expression of the gene for ribosomal protein S20: effects of gene dosage.

We have exploited the properties of three different plasmids which carry the gene for Escherichia coli ribosomal protein S20 (rpsT) to test the effects of gene dosage on the expression of rpsT. Over a range of total copies of rpsT of 1 to 58 per haploid genome equivalent, the rate of incorporation of uridine during a 30-s pulse into RNA annealing to either of two specific probes for S20 mRNA increased essentially in proportion to copy number. In contrast, the rate of synthesis of S20 protein increased no more than 2.1-fold at the highest copy number. We conclude, in contrast to an earlier report (D. Geyl, and A. Böck, Mol. Gen. Genet. 154:327-334, 1977), that the synthesis of S20 is regulated at a posttranscriptional step. We propose that S20 itself is the regulatory agent and that binding of S20 to its own mRNA in regions homologous in structure with 16S rRNA can account for our results.