Protein synthesis in rabbit reticulocytes. XIII. Lack of mRNA (poly r (A)) binding activity in highly purified EIF-1.

Met-tRNA_f^Met binding factor (EIF-1) has been purified more than 100 fold over crude high salt (0.5 M KCl) ribosomal wash. The purified factor binds 2 nmoles Met-tRNA_f^Met per mg protein and shows very little poly r(A) binding activity. Crude ribosomal high salt wash possesses significant amounts of poly r(A) binding activity and also binds to other RNAs. The bulk of this unspecific RNA binding protein is separated from EIF-1 by DEAE-cellulose chromatography.     

Cloning of cDNA sequences for an Artemia salina hnRNP protein: evidence for conservation through evolution.

A cDNA clone was isolated for Artemia salina protein HD40, a component of heterogenous nuclear ribonucleoproteins. Enriched Artemia 15S poly(A)+ RNA was used as a template and double-stranded cDNA sequences were inserted into the Pst I restriction endonuclease site of E. coli plasmid pBR322. Recombinant colonies were analyzed by positive hybrid selection of poly(A)+ RNA that directs the synthesis of protein HD40 in an in vitro assay. In vitro translation of the mRNA selected by recombinant clone 87HD yields a protein that is immunoprecipitated by anti-HD40 antibodies and that comigrates with authentic HD40 on gel electrophoresis. Partial proteolysis of protein HD40 and the in vitro translated product selected by clone 87HD produces the same peptide patterns. The size of the cloned insert is about 820 bp. The length of HD40 mRNA as determined by Northern blot analysis, is about 1500 nucleotides. Southern blot analysis performed with DNA of different species (plant, avian, mammal) shows cross-hybridizing bands when probed with clone 87HD DNA suggesting that the HD40 gene is evolutionarily conserved.     

A single-stranded nucleic acid-binding protein from Artemia salina. II. Interaction with nucleic acids

A helix-destabilizing protein, HD40 (Mr 40,000), isolated from the cytoplasm of Artemia salina (Marvil, D.K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466-6472) stoichiometrically disrupts the secondary structures of synthetic single-stranded and helical polynucleotides (e.g. poly(rA), poly(dA), poly(rC), poly(dC), and poly(rU)) as well as those of natural polynucleotides (e.g. MS2 RNA and phi X174 viral DNA). The conformations of double-stranded DNA and double- or triple-stranded synthetic polynucleotides are not affected by the protein. Formation of duplexes, e.g. poly(rA . rU), is prevented by HD40 at 25 to 50 mM but not at 100 to 140 mM NaCl. The unwinding of the residual secondary structure of RNA and DNA by HD40 is not highly cooperative and has a stoichiometry of one HD40 per 12 to 15 nucleotides. The addition of HD40 in excess of 1 molecule per 12 to 15 nucleotides results in the cooperative formation of distinct bead-like structures along the nucleic acid strand. The beads are about 20 nm in diameter with a center to center distance of about 40 nm. The appearance of the beads is not accompanied by any spectral changes (CD and UV) beyond those obtained at a stoichiometry of one HD40 molecule per 12 to 15 nucleotides.     

Structure of complexes between a major protein of heterogeneous nuclear ribonucleoprotein particles and polyribonucleotides

We have investigated the structure of complexes formed between a series of poly(A)n (n = 30 to 480) and HD40 (helix-destabilizing protein, molecular weight of 40,000), the major protein component of 30 S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from the brine shrimp Artemia salina. Protein HD40 is similar to corresponding hnRNP proteins from higher eukaryotes and the complexes it forms with single-stranded nucleic acids are strikingly similar to the native "beads-on-a-string" structure of hnRNP. Using analytical ultracentrifugation and electron microscopy we find: (1) complexes formed between HD40 and long ribohomopolymers also have a beads-on-a-string structure, showing that the ability to form this structure is an inherent property of HD40, and is not dependent on any structural features of natural RNA; (2) complexes between HD40 and poly(A)160 form disks that are about 3 nm high by 18 nm in diameter and contain 20 HD40 molecules; (3) complexes of HD40 with poly(A)n with fewer than 160 nucleotides form sectors of a disk: 40 nucleotides give rise to a quarter of a disk, 80 nucleotides, half a disk, etc. The molecular weights increase with the size of poly(A)n at the rate of 5300 per nucleotide, a stoichiometry of eight nucleotides per HD40; (4) as the size of the poly(A)n increases beyond 160 nucleotides, the additional nucleoprotein elements may either initiate the formation of a second disk adjacent to the first or stack on top of the first disk to form a 6 nm high helix with a diameter of 18 nm. Based on these results, we propose that the existence of lateral protein-protein interactions that produce the basic 3 nm X 18 nm disk, combined with the marginal stability of the helix result in (a) interruptions of the helix that give rise to the beads-on-a-string appearance of the complexes, and (b) inherent heterogeneity of individual "beads" which may contain one or more turns of the helix. From measurements of HD40 complexes with coliphage MS2 RNA, phi X174 viral DNA as well as with the homopolymers, a bead is estimated to contain an average of approximately 300 nucleotides; approximately 1 X 8 turns of the helix.     

Cloning of a full-length complementary DNA for an Artemia salina glycine-rich protein. Structural relationship with RNA binding proteins

Overlapping cDNAs have been isolated containing all the coding sequences for Artemia salina protein GRP33, a glycine-rich protein (16.6 mol % glycine), with a molecular weight of 32,992. GRP33 is closely related to HD40, the major protein component of Artemia heterogeneous nuclear ribonucleoprotein particles, and shares certain characteristics with other RNA binding proteins. The C-terminal region (123 amino acids) contains 39 glycine residues. This region has multiple arginine residues flanked by glycines, resembling the glycine-dimethylarginine clusters present in other RNA binding proteins. Secondary structure predictions for the protein reveal two distinct domains: a hydrophilic C-terminal domain with an extended conformation and a larger N-terminal domain with a number of alpha-helices and beta-sheets.     

A cell-free mammalian protein-synthesizing system stimulated by RNA from avian myeloblastosis virus.

Carcinoma cells, oncornavirus-infected cells and fetal bovine tissue provide salt wash ribosomal factors capable of responding to avian myeloblastosis virus (AM virus)-RNA and stimulating the incorporation of amino acids into proteins as well as catalyzing the binding of N-acetylated (35S) methionyl-tRNA. The exogenously dependent amino acid incorporation system is stimulated by the high molecular weight species of AM virus-RNA only, particularly the fraction containing polyadenylate (poly(A)) residues; the system is also markedly inhibited by the low molecular weight AM virus-RNA species. Activity for the exogenous system displays very definite divalent/monovalent cation optima and requires the presence of mammalian transfer RNA.     

Human elongation factor 1 beta: cDNA and derived amino acid sequence.

From a cDNA library in lambda gt11 derived from poly (A+)RNA of human ovarian granulosa cells a cDNA clone lambda HGP34, containing an EcoRI insert of 829 bp, was identified. After subcloning of the insert into pUC18, the clone pHGP34 was obtained and sequenced. The derived amino acid sequence, corresponding to a protein of 225 amino acids, shows a high degree of homology to elongation factor 1 beta (EF-1 beta) of Artemia salina (57%) and known peptide sequences of Xenopus laevis EF-1 beta (86%). We therefore assume that the protein coded for by pHGP34 represents human EF-1 beta. Northern analysis reveals an EF-1 beta specific mRNA of 900 bp. Southern analysis indicates that EF-1 beta in the human genome, like EF-1 alpha, appears to be specified by more than one gene. A high degree of sequence homology for EF-1 beta specific sequences is observed for bovine, rat and mouse species.     

The expression of a gene for eukaryotic elongation factor Tu in Artemia during development. Translation of poly(A)+ RNA and the use of a synthetic oligonucleotide to detect the presence of eukaryotic elongation factor Tu-specific mRNA

Total in vivo proteins from Artemia embryos at different developmental stages were examined by two-dimensional gel electrophoresis. A variety of peptides change during development, with one of them, the eukaryotic elongation factor Tu (eEF-Tu), presenting a dramatic increase from dormant embryos to nauplii. When poly(A)+ RNA is translated in vitro, the same relative increase is seen for eEF-Tu during development. Based on the amino acid sequence for Artemia eEF-Tu (Amons, R., Pluijms, W., Roobol, K., and M?ller, W. (1983) FEBS Lett. 153, 37-42), a synthetic oligodeoxynucleotide was prepared and used to prime the synthesis of cDNA with poly(A)+ RNA from 12-h developing embryos as template. Direct sequence analysis of the 900-base primary cDNA product shows it to be specific for the 5' end of Artemia eEF-Tu mRNA. Hybridization of a "Northern" blot of denatured (poly(A)+ RNA from different developmental stages with this cDNA reveals a major band migrating at about 1800 bases, which increase in intensity as development proceeds, paralleling the increase in eEF-Tu seen by in vitro translation. When poly(A)+ RNA is separated on a nondenaturing gel, blotted to poly(U) paper, and hybridized with the eEF-Tu cDNA, a single band is observed migrating faster than 18 S. Elution and in vitro translation of this band results in a major product migrating with eEF-Tu in a dodecyl sulfate-polyacrylamide gel and which is precipitable with eEF-Tu-specific antibodies.     

Nucleic acid binding and unfolding properties of ribosomal protein S1 and the derivatives S1-F1 and m1-S1.

The nucleic acid binding and unwinding properties of wild-type Escherichia coli ribosomal protein S1 have been compared to those of a mutant form and a large trypsin-resistant fragment, both reported recently [J. Mol. Biol. 127, 41-45 (1979) and J. Biol. Chem. 254, 4309-4312 (1979). The mutant (m1-S1) contains 77% and the fragment (S1-F1) 66% of the polypeptide chain length (approximately 600 amino acid residues) of protein S1. The mutant is active in protein synthesis in vitro; the fragment, although retaining one or more of the functional domains of S1, is inactive in protein synthesis. We find that m1-S1 is is almost as effective as S1 in binding to poly(rU), phage MS2 RNA and simian virus 40 (SV40) DNA, and in unfolding poly(rU) and the helical structures present in MS2 RNA and phi X174 viral DNA. S1-F1, however, binds to poly(rU) and denatured SV40 DNA, but not to MS2 RNA. It unfolds neither poly(rU), nor the residual secondary structure of MS2 RNA or phi X174 viral DNA. Thus, there appears to be a correlation between the loss in ability of S1 to unwind RNA and the loss in its ability to function in protein synthesis.     

The synthesis of RNA and silk protein in Galleria mellonella silk glands

The silk protein synthesis in silk glands of Galleria mellonella is preceded by the increase of total RNA content. The levels of the main RNA classes: 28S and 18S rRNA, tRNA as well as poly(A) + RNA change proportionally to the total RNA pool of glands. The fibroin-like silk protein of molecular weight of about 240 000 is characterized by the high content of four amino acids: glycine, alanine, serine and leucine, which account for more than 70% of amino acid residues. This fibroin-like protein is present in the posterior, middle and anterior parts of silk gland of the last instar larvae.     

Characterization of a poly(A)+RNA-containing structural component directly associated with cytoplasmic membranes in dormant Artemia cysts

Poly(A)+RNA-containing material was extracted from the purified cytoplasmic membranes of dormant Artemia cysts by treatment with mild detergents. Sedimentation analysis of the extracts showed a predominant poly(A)-containing fraction at 40 S, associated with about 6% of the extracted proteins. Only limited amounts of poly(A)-containing material were found in the heavier fractions. Poly(A)+RNA extracted from the 40-S fraction sedimented around 14 S. The poly(A)-containing 40-S structures could be purified by treatment with non-ionic or zwitterionic detergents followed by resedimentation in sucrose gradients in the presence or absence of detergent. When the 40-S fraction was analyzed by isopycnic centrifugation in Cs2SO4 gradients, the main part of the poly(A)-containing material banded at a density of 1.27 g/ml. Electron-microscopic examination of this fraction revealed circular or slightly bullet-shaped profiles measuring 17-26 nm. When the 40-S fraction had been submitted to mild RNAase treatment prior to density gradient centrifugation, the material was displaced towards lower density and became less distinct. Purified 40-S particles showed a complex protein pattern not very similar to that of polyribosomal poly(A)+RNA-containing particles from developing embryos, but with components in common with unfractionated membranes. The particles also contained some lipids. The experiments indicate that a major part of the membrane-bound, latent poly(A)+RNA in dormant Artemia cysts occurs in the form of relatively uniform, detergent- and Cs2SO4-resistant structures, independent of ribosomes, but intimately associated with membrane components.     

RNA binding properties of the coat protein from bacteriophage GA.

The coat protein of bacteriophage GA, a group II RNA phage, binds to a small RNA hairpin corresponding to its replicase operator. Binding is specific, with a Ka of 71 microM -1. This interaction differs kinetically from the analogous coat protein-RNA hairpin interactions of other RNA phage and also deviates somewhat in its pH and salt dependence. Despite 46 of 129 amino acid differences between the GA and group I phage R17 coat proteins, the binding sites are fairly similar. The essential features of the GA coat protein binding site are a based-paired stem with an unpaired purine and a four nucleotide loop having an A at position -4 and a purine at -7. Unlike the group I phage proteins, the GA coat protein does not distinguish between two alternate positions for the unpaired purine and does not show high specificity for a pyrimidine at position -5 of the loop.     

Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.     

Nonselective inhibition of messenger RNA translation by highly purified low molecular weight RNA species from ribosomal salt wash of chick embryonic muscle

A low molecular weight RNA species, in the 70–90 nucleotide size range (iRNA), has been purified from the ribosomal salt wash of chick embryonic muscle by a combination of DEAE-cellulose and hydroxyapatite chromatography. This method yields iRNA free from contaminating tRNA and gives better and more reproducible yields than those obtained with our previous method involving lengthy dialysis of the salt wash. The iRNA at a concentration of 20–80 ng range strongly inhibits the translation of homologous and heterologous mRNAs i.e. chick muscle poly(A)⁺mRNA and rabbit globin mRNA; uncapped mRNA; and poly(A)^-mRNA in micrococcal nuclease-treated reticulocyte lysate indicating that inhibition by iRNA is nonselective in nature. The translation of endogenous globin mRNA and polysomes in the lysate is strikingly less sensitive to iRNA suggesting that the initiation step is primarily affected by iRNA. The iRNA does not appear to be double-stranded RNA. It is concluded that iRNA is distinct from other low molecular weight RNA species described in the literature which modulate protein synthesis in cell-free systems.     

A fluorescence study of the binding of poly(1,N6-ethenoadenylic acid) to Escherichia coli initiation factor 3

The binding of initiation Factor 3 (IF3) to poly (1,N6-ethenoadenylic acid) [poly(epsilon A)] was investigated by fluorescence spectroscopy. At low salt concentrations, IF3 evokes an increase in the fluorescence intensity of poly(epsilon A) due to the unstacking of the nucleotide bases. The poly(epsilon A) fluorescence enhancement titrates to an endpoint of 13 +/- 2 nucleotide residues per IF3. The maximum poly(epsilon A) fluorescence enhancement, at lattice saturation, decreases with increasing salt concentration. Even though IF3 does not produce a large fluorescence increase between 75 and 200 mM NaCl concentration, the protein still binds to poly(epsilon A) at these salt concentrations as measured by sedimentation partition chromatography; the value of Kobs for the IF3-poly(epsilon A) interaction is comparable to that of other synthetic polynucleotides. The binding of IF3 to poly(A) at 150 and 200 mM NaCl induces an increase in nucleotide base-base separation as determined by CD, yet IF3-induced disruption of base stacking of poly(epsilon A) at these same salt concentrations is not detected by fluorescence. It is likely that IF3 binds primarily to the phosphate backbone of poly(epsilon A) at low salt concentrations, producing an increase in the fluorescence intensity. But, at higher salt concentrations, the aromatic amino acids intercalate between the nucleotide bases quenching the poly(epsilon A) fluorescence.     

Characterization and messenger activity of poly(A)-containing RNA from Chlorella.

Poly(A)-containing RNA was isolated by cellulose column chromatography from total RNA extracted from Chlorella fusca var. vacuolata 211/8p. RNA retained by the column was identified as poly(A)-containing RNA because it contained ribonuclease-resistant tracts, 25 to 55 nucleotides in length, from which not less than 80% of base was found to be adenine after acid hydrolysis. The base composition of poly(A)-containing RNA differed from that of RNA (largely ribosomal) which did not adsorb to cellulose, having a higher adenine content and a lower guanine content. Poly(A)-containing RNA was polydisperse including molecules with mobilities from 10S to 40S with a mean of about 20S. In an in vitro system derived from wheat-germ, protein synthesis was stimulated by adding poly(A)-containing RNA from Chlorella. Optimum conditions were established in this system with respect to the amount of poly(A)-containing RNA added and the concentration of KCl and Mg-2+. It is proposed that, in Chlorella, poly(A)-containing RNA includes cytoplasmic mRNA as has been shown for some other eucaryotic organisms.     

Requirement of chain initiation factor 3 and ribosomal protein S1 in translation of synthetic and natural messenger RNA.

Amino acid incorporation directed by poly(A), poly(U) or R17 RNA has been examined in S1-depleted protein synthesizing systems. We observe that the translation of either synthetic or natural messenger RNA is strictly dependent on the presence of chain initiation factor 3 and ribosomal protein S1. With poly(A) or poly(U) both IF-3 and S1 stimulate amino acid incorporation at least 25-fold, and with R17 RNA the stimulation is approximately 15-fold. More than one copy of S1 per ribosome decreases amino acid incorporation directed by poly(U) or R17 RNA. Initiation complex formation with R17 RNA is also stimulated optimally by the addition of one copy of S1 per ribosome. The function of IF-3 and S1 in protein synthesis is considered.     

Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity.     

The MDM2 oncoprotein binds specifically to RNA through its RING finger domain.

BACKGROUND: The cellular mdm2 gene has transforming activity when overexpressed and is amplified in a variety of human tumors. At least part of the transforming ability of the MDM2 protein is due to binding and inactivating the p53 tumor suppressor protein. Additionally, this protein forms a complex in vivo with the L5 ribosomal protein and its associated 5S ribosomal RNA and may be part of a ribosomal complex. MATERIALS AND METHODS: A RNA homopolymer binding assay and a SELEX procedure have been used to characterize the RNA-binding activity of MDM2. RESULTS: The MDM2 protein binds efficiently to the homopolyribonucleotide poly(G) but not to other homopolyribonucleotides. This binding is independent of the interaction of MDM2 with the L5 protein, which occurs through the central acidic domain of MDM2. An RNA SELEX procedure was performed to identify specific RNA ligands that bind with high affinity to the human MDM2 (HDM2) protein. After 10 rounds of selection and amplification, a subset of RNA molecules that bound efficiently to HDM2 was isolated from a randomized pool. Sequencing of these selected ligands revealed that a small number of sequence motifs were selected. The specific RNA binding occurs through the RING finger domain of the protein. Furthermore, a single amino acid substitution in the RING finger domain, G446S, completely abolishes the specific RNA binding. CONCLUSIONS: These observations, showing that MDM2 binds the L5/5S ribosomal ribonucleoprotein particle and can also bind to specific RNA sequences or structures, suggest a role for MDM2 in translational regulation in a cell.