Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein.

The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides upstream of the polyadenylate tail are conserved in all genes. There is no equivalent of the eucaryotic polyadenylation signal AAUAAA upstream of the polyadenylate tail. The matrix protein of 28,717 daltons has 256 amino acids. It is relatively basic and moderately hydrophobic. There are two clusters of hydrophobic amino acid residues in the C-terminal third of the protein that could potentially interact with the membrane components of the infected cell. The matrix protein has no homology with the matrix proteins of other negative-strand RNA viruses, implying that RS virus has undergone extensive evolutionary divergence. A second open reading frame potentially encoding a protein of 75 amino acids and partially overlapping the C terminus of the matrix protein was also identified.     

Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F.

The mechanism of ribosome binding to eucaryotic mRNAs is not well understood, but it requires the participation of eucaryotic initiation factors eIF-4A, eIF-4B, and eIF-4F and the hydrolysis of ATP. Evidence has accumulated in support of a model in which these initiation factors function to unwind the 5'-proximal secondary structure in mRNA to facilitate ribosome binding. To obtain direct evidence for initiation factor-mediated RNA unwinding, we developed a simple assay to determine RNA helicase activity, and we show that eIF-4A or eIF-4F, in combination with eIF-4B, exhibits helicase activity. A striking and unprecedented feature of this activity is that it functions in a bidirectional manner. Thus, unwinding can occur either in the 5'-to-3' or 3'-to-5' direction. Unwinding in the 5'-to-3' direction by eIF-4F (the cap-binding protein complex), in conjunction with eIF-4B, was stimulated by the presence of the RNA 5' cap structure, whereas unwinding in the 3'-to-5' direction was completely cap independent. These results are discussed with respect to cap-dependent versus cap-independent mechanisms of ribosome binding to eucaryotic mRNAs.     

A Saccharomyces cerevisiae homologue of mammalian translation initiation factor 4B contributes to RNA helicase activity.

The TIF3 gene of Saccharomyces cerevisiae was cloned and sequenced. The deduced amino acid sequence shows 26% identity with the sequence of mammalian translation initiation factor eIF-4B. The TIF3 gene is not essential for growth; however, its disruption results in a slow growth and cold-sensitive phenotype. In vitro translation of total yeast RNA in an extract from a TIF3 gene-disrupted strain is reduced compared with a wild-type extract. The translational defect is more pronounced at lower temperatures and can be corrected by the addition of wild-type extract or mammalian eIF-4B, but not by addition of mutant extract. In vivo translation of beta-galactosidase reporter mRNA with varying degree of RNA secondary structure in the 5' leader region in a TIF3 gene-disrupted strain shows preferential inhibition of translation of mRNA with more stable secondary structure. This indicates that Tif3 protein is an RNA helicase or contributes to RNA helicase activity in vivo.     

The 5'-end structure of ovalbumin mRNA in isolated nuclei and polysomes.

We used the chemical reagents dimethylsulfate and 4'-aminomethyl-4,5',8-trimethylpsoralen and the enzyme T1 ribonuclease to compare the 5'-end structure of ovalbumin mRNA in situ in purified hen oviduct nuclei and polysomes with that of the isolated mRNA. The qualitative pattern of structure-dependent base modifications and T1 ribonuclease cleavage sites in intranuclear and polysomal ovalbumin mRNAs was found to be nearly identical to those in isolated ovalbumin mRNA. These structural data are consistent with the presence of a trigonal stem-loop structure at the 5'-end of ovalbumin mRNA (hairpin-1) in nuclei and polysomes. Similar results were obtained for a coding region structure (hairpin-3) in intranuclear ovalbumin mRNA. We have recently shown that hairpin-1 positively affects the rate of ovalbumin mRNA translation in vitro and is part of a high affinity binding site for eucaryotic initiation factor-2 (eIF-2). The presence of hairpin-1 in ovalbumin mRNA in both a pretranslation state (nuclei) and active translation state (polysomes) is consistent with its hypothesized biological function as an intracellular initiation signal that facilitates the translation of this mRNA.     

Functional and Structural Analysis of Maize Hsp101 IRES

Maize heat shock protein of 101 KDa (HSP101) is essential for thermotolerance induction in this plant. The mRNA encoding this protein harbors an IRES element in the 5′UTR that mediates cap-independent translation initiation. In the current work it is demonstrated that hsp101 IRES comprises the entire 5′UTR sequence (150 nts), since deletion of 17 nucleotides from the 5′ end decreased translation efficiency by 87% compared to the control sequence. RNA structure analysis of maize hsp101 IRES revealed the presence of three stem-loops toward its 5′ end, whereas the remainder sequence contains a great proportion of unpaired nucleotides. Furthermore, HSP90 protein was identified by mass spectrometry as the protein preferentially associated with the maize hsp101 IRES. In addition, it has been found that eIFiso4G rather than eIF4G initiation factor mediates translation of the maize hsp101 mRNA.     

Effect of mutations and deletions in a bicistronic mRNA on the synthesis of influenza B virus NB and NA glycoproteins.

The mRNA derived from influenza B virus RNA segment 6 is functionally bicistronic and encodes the NB and NA glycoproteins in different, overlapping reading frames. NB protein synthesis is initiated at the 5'-proximal AUG codon, and 4 nucleotides downstream there is a second AUG codon which is used to initiate NA protein synthesis. The nucleotide sequence context of the first AUG codon conforms closely with the established 5'-CC(A/G)CCAUGG-3' consensus sequence (M. Kozak, Nucleic Acids Res. 15:8125-8148, 1987), which should favor initiation of NB protein synthesis at this site, yet NB and NA are found to accumulate in approximately equal amounts in infected cells. To determine the features important for allowing initiation at the second 5'-proximal AUG codon, we made changes in the 5'-terminal region of the mRNA, including deletions, insertions, and site-specific mutations. The recombinant DNA molecules were expressed in eucaryotic cells, and the accumulation of NB and NA was quantitated. The data indicate that changes in the immediate sequence around the first AUG codon do not make a large difference in the amounts of NB and NA that accumulate, but that when the first AUG codon is displaced from its normal position it is now quite efficient at preventing downstream initiation events. In addition, the data indicate that an element of the B/NB/NA mRNA 5' untranslated leader region acts in cis to enhance the expression of NB and NA.     

Eucaryotic initiation factor 4B of wheat germ binds to the translation initiation region of a messenger ribonucleic acid

Purified preparations of eucaryotic initiation factor 4B (eIF4B) from wheat germ bind the monocistronic, uncapped, mRNA satellite tobacco necrosis virus RNA (STNV RNA) in nitrocellulose-mediated binding assays. This reaction is mRNA specific and yields dissociation constants (Kd) in the 10(-7)-10(-8) M range, depending upon the particular enzyme preparation tested. Purified wheat germ eIF4A, in the presence or absence of ATP, does not bind STNV RNA efficiently, but added eIF4A and ATP do enhance the efficiency of the eIF4B-dependent binding of STNV RNA. Wheat germ eIF4B binds the oligonucleotide containing the 5'-terminal 52 nucleotides of STNV RNA (designated 1-52) with the same affinity as intact STNV RNA. This binding affinity is less with the 1-44 oligonucleotide of STNV RNA and does not occur with the 1-33 oligonucleotide of STNV RNA that contains the 5'-terminal untranslated region and the initiator AUG codon at positions 30-32 of this mRNA. Wheat germ eIF4B therefore binds the translation initiation region of STNV RNA, and this binding requires up to 20 nucleotides on the 3' side of the initiator AUG codon of this mRNA. Wheat germ eIF4B also efficiently binds an oligonucleotide containing nucleotides from positions 13-52 in from the 5' terminus of STNV RNA, thereby establishing that the postulated 5'-terminal stem and loop secondary structure of STNV RNA [Leung, D. W., Browning, K. S., Heckman, J. E., RajBhandary, U. L., & Clark, J. M., Jr. (1979) Biochemistry 18, 1361-1366] is not functional or essential for this specific binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel low molecular weight ribonucleic acid (RNA) related to transforming growth factor alpha messenger RNA

Human transforming growth factor alpha (TGF alpha) is coded for by an mRNA of about 4800 nucleotides. The cDNA sequence demonstrates that the 50 amino acid TGF alpha is embedded in a larger 160 amino acid precursor protein. We report here that in addition to the 4800 nucleotide TGF alpha mRNA, there is a novel second RNA species of about 350 nucleotides that hybridizes to a human TGF alpha cDNA probe. This small RNA species has been found in the RNA of several human tumor cells including HT1080, A549, A431, A2058, and A673. We have demonstrated an inverse relationship between the amounts of the 4800 nucleotide TGF alpha mRNA and the 350 nucleotide novel RNA in these human cells. Restriction enzyme cleavage of a human TGF alpha cDNA probe into three separate domains consisting of a processed coding region and 5'- and 3'-preprocessed coding and untranslated regions showed that only the 3'-untranslated region hybridized to the 350 nucleotide RNA. Using sense and anti-sense single-stranded 3'-untranslated region probes, we determined that the 350 nucleotide RNA band may be composed of multiple species of RNA which are related to the anti-sense DNA strand that is opposite to the strand that codes for the 4800 nucleotide TGF alpha mRNA.     

Nucleotide sequences from the 3''-ends of vesicular stomatitis virus mRNA''s as determined from cloned DNA.

Molecular clones of vesicular stomatitis virus mRNA's were used to determine the 3'-terminal sequences of mRNA's encoding the N and NS proteins. This new approach to VSV mRNA sequencing allowed the first comparison of 3'-terminal sequences. The sequences showed a tetranucleotide homology, UAUG, immediately preceding the polyadenylic acid. In addition, both mRNA's had an AU-rich region including the tetranucleotide AUAU at positions 16 to 19 nucleotides from the polyadenylic acid. A possible secondary structure between the 3' end of N mRNA and the 5' end of the adjacent NS mRNA is noted. These structural features may serve as signals for termination (or cleavage) and polyadenylation of vesicular stomatitis virus mRNA's. Neither mRNA had the polyadenylic acidproximal hexanucleotide, AAUAAA, found in eucaryotic cellular and viral mRNA's transcribed from nuclear DNA. The probable location of the translation termination codon for the NS protein is only six nucleotides from polyadenylic acid in NS mRNA.     

Messenger RNA structure participating in the initiation of synthesis of cucumber mosaic virus coat protein

The sequence of the 5'-terminal 106 nucleotides of cucumber mosaic virus (strain Y) RNA 4, the mRNA coding for viral coat protein, has been determined. The first AUG was located at 77 nucleotides from the 5'-terminus and was confirmed to be an initiation codon by analysis of the N-terminal amino acid sequence of the protein. The nucleotide sequence (positions 77-106) beyond the AUG codon predicted the sequence of ten amino acids corresponding to the N-terminal region of the protein, which exactly matched the determined amino acid sequence containing an acetyl methionine as the N-terminal amino acid. The distance of the initiation codon AUG from the cap structure was 76 nucleotides and the longest among the mRNAs for coat protein of plant viruses so far reported (9-36 nucleotides). This noncoding region is rich in U residues (40%) and the number of G residues (21 nucleotides) is the largest among these mRNAs (usually 1 or 2 residues). A possible secondary structure is postulated for the region, which might be implicated in efficient translation of the RNA 4 in vivo.     

Insulin induction of ornithine decarboxylase. Importance of mRNA secondary structure and phosphorylation of eucaryotic initiation factors eIF-4B and eIF-4E

We investigated the possibility that insulin could stimulate translation of ornithine decarboxylase (ODC) mRNA in a murine fibroblast cell line that expresses large numbers of human insulin receptors (HIR 3.5 cells). Within 3 h after exposure to 70 nM insulin, ODC enzyme activity increased approximately 50-fold and mRNA accumulation 3-fold in the HIR 3.5 cells but not in normal fibroblasts. Pretreatment of cells with cycloheximide completely inhibited insulin-stimulated ODC expression; actinomycin D partially inhibited this effect. To determine the influence of the 5' untranslated region (5'UTR) of ODC mRNA on insulin-regulated ODC expression, plasmids were constructed which contained sequences from the 5'UTR of a rat ODC mRNA interposed between the ferritin promoter and the coding region of the human growth hormone gene. These constructions were then expressed transiently in HIR 3.5 cells. Insulin stimulated a 2-4-fold change in growth hormone accumulation in the medium of cells transiently expressing plasmids containing the entire 5'UTR of ODC mRNA or just the 5'-most 115 bases, a G/C-rich conserved sequence predicted to form a stem-loop structure and shown previously to be responsible for constitutive inhibition of translation. There was a direct correlation between the extent of insulin stimulation and the predicted secondary structure of the added 5'UTR sequences. To determine whether this effect might be due to insulin activation of initiation factors responsible for melting mRNA secondary structure, we examined the effect of insulin on the phosphorylation states of two such factors, eucaryotic initiation factors eIF-4B and eIF-4E. Insulin stimulated the phosphorylation of both initiation factors; this stimulation was evident at 15 min and maximal by 60 min. These results suggest a potential general mechanism by which insulin could preferentially stimulate translation of mRNAs whose 5'UTRs exhibit significant secondary structure by activating initiation factors involved in melting such secondary structures.     

A heat-sensitive inhibitor in poliovirus-infected cells which selectively blocks phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the double-stranded RNA-activated protein kinase.

We partially purified an inhibitor from poliovirus-infected HeLa cells which specifically blocked phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the double-stranded RNA-activated protein kinase. The inhibitory activity eluted from a sizing column with an approximate molecular weight of 80,000 to 100,000 and was sensitive to heat, suggesting a protein nature for the inhibitor. No specific virus-encoded protein purified with the inhibitor. The inhibition of phosphorylation of the alpha subunit of eucaryotic initiation factor 2 was not due to a protein phosphatase associated with the inhibitor. The inhibitor did not seem to prevent phosphorylation of the double-stranded RNA-activated protein kinase but inhibited the phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the activated kinase. Double-stranded RNA-induced inhibition of in vitro protein synthesis in reticulocyte lysates could be prevented by the addition of the partially purified inhibitor during preincubation of lysate with double-stranded RNA.     

Complete cDNA sequence for rabbit muscle glycogen phosphorylase

The cDNA for the nearly full-length rabbit muscle glycogen phosphorylase mRNA has been isolated and sequenced. The cDNA is rich in G and C nucleotides. This feature is especially striking at the 3rd position of codons, where 86% of the 843 amino acid codons terminate with G or C. Methionine, presumably the initiation residue, is found at position-1, suggesting that the removal of only a single methionine residue precedes the amino-terminal acetylation at serine. Eight differences between the deduced amino acid sequence and the previously determined protein sequence are discussed.