Elongation Step Of Protein Synthesis Research Articles

Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A.

Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion/suppression and by the activation of p53 and Siah-1. To better characterize the function of TCTP, a yeast two-hybrid hunt was performed. Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1Bbeta, as TCTP-interacting partners. In vitro and in vivo studies confirmed that TCTP bound specifically eEF1Bbeta and eEF1A. Additionally, MS analysis also identified eEF1A as a TCTP interactor. Because eEF1A is a GTPase, we investigated the role of TCTP on the nucleotide exchange reaction of eEF1A. Our results show that TCTP preferentially stabilized the GDP form of eEF1A, and, furthermore, impaired the GDP exchange reaction promoted by eEF1Bbeta. These data suggest that TCTP has guanine nucleotide dissociation inhibitor activity, and, moreover, implicate TCTP in the elongation step of protein synthesis.

Kinectin Anchors the Translation Elongation Factor-1δ to the Endoplasmic Reticulum

Kinectin has been proposed to be a membrane anchor for kinesin on intracellular organelles. A kinectin isoform that lacks a major portion of the kinesin-binding domain does not bind kinesin but interacts with another resident of the endoplasmic reticulum, the translation elongation factor-1 delta (EF-1 delta). This was shown by yeast two-hybrid analysis and a number of in vitro and in vivo assays. EF-1 delta provides the guanine nucleotide exchange activities on EF-1 alpha during elongation step of protein synthesis. The minimal EF-1 delta-binding domain on kinectin resides within a conserved region present in all the kinectin isoforms. Overexpression of the kinectin fragments in vivo disrupted the intracellular localization of EF-1 delta proteins. This report provides evidence of an alternative kinectin function as the membrane anchor for EF-1 delta on the endoplasmic reticulum and provides clues to the EF-1 complex assembly and anchorage on the endoplasmic reticulum.

Adenylyl cyclase-dependent form of chemical long-term potentiation triggers translational regulation at the elongation step

The persistent maintenance of long-term potentiation requires both messenger RNA and protein synthesis. While there is mounting evidence for an active role of protein synthesis in hippocampal long-term potentiation, the nature of mechanisms underlying its regulation has not yet been established. We used a previously described chemical long-term potentiation protocol [J Neurosci 19 (1999) 2500] to address the hypothesis that signaling mechanisms, involved in long-lasting long-term potentiation, directly regulate protein synthesis. Chemical long-term potentiation is an N-methyl- d-aspartate receptor-dependent form of plasticity, which relies on both synaptic activity, in the form of spontaneous bursting induced by high concentrations of K + and Ca 2+, and cyclic AMP/adenylyl cyclase signaling. We found that chemical long-term potentiation in CA1 of the mouse hippocampus lasts for at least 3 hours and requires both messenger RNA and protein synthesis. However, surprisingly de novo total protein synthesis was paradoxically decreased at 1 hour after long-term potentiation induction. Consistent with the decrease in total protein synthesis in potentiated CA1, phosphorylation of eukaryotic elongation factor 2 was increased and is likely responsible for inhibition of translation at the elongation step. Increased phosphorylation of eukaryotic elongation factor 2 was dependent on coincident cyclic AMP/adenylyl cyclase activation and synaptic activity and required N-methyl- d-aspartate receptor activation. Despite the inhibition in total protein synthesis, the level of the immediate early gene protein Arc (activity regulated cytoskeleton-associated protein) increased at 1 hour after chemical long-term potentiation induction. Taken together, the results suggest that regulation at the elongation step of protein synthesis contributes to persistent forms of long-term potentiation.

N-Methyl- D-aspartate receptor activation inhibits protein synthesis in cortical neurons independently of its ionic permeability properties

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate, strongly depresses protein synthesis. We have previously demonstrated in cortical neurons that a glutamate-induced increase in intracellular Ca 2+ is likely responsible for the blockade of the elongation step of protein synthesis. In this study, we provide evidence indicating that NMDA mobilizes a thapsigargin-sensitive pool of intracellular Ca 2+. Exposure of cortical neurons to NMDA, in the absence of external Ca 2+, produced a transient rise in intracellular Ca 2+ that was suppressed by pretreatment with thapsigargin. This rise in intracellular Ca 2+ did not result from an influx of Na + via reversal of the mitochondrial Na +/Ca 2+ exchanger since it persisted in a Na +-free medium or in the presence of CGP 37157, an inhibitor of the exchanger. Moreover, the NMDA-induced increase in intracellular Ca 2+ required the presence of D-serine, was blocked by D(−)-2-amino-5-phosphonopentanoic acid, but was not reduced in the presence of external Mg 2+. This unexpected non-ionotropic effect of NMDA was associated with an inhibition of protein synthesis that was also insensitive to the absence of external Ca 2+ or Na +, or presence of Mg 2+. NMDA treatment resulted in an increase in the phosphorylation of eEF-2 in the absence or presence of external Ca 2+. The initiation step of protein synthesis was not blocked by NMDA since the phosphorylation of initiation factor eIF-2α subunit was not altered by NMDA treatment. In conclusion, we provide evidence indicating that NMDA can inhibit protein synthesis in cortical neurons through a process that involves the mobilization of intracellular Ca 2+ stores via a mechanism that is not linked to the ionic properties of NMDA receptors.

Cultured Aedes albopictus mosquito cells accumulate elongation factor-1α (EF-1α) during serum starvation

We examined survival, growth and protein synthesis in mosquito cells that had been maintained for up to 21 days in serum-free medium. On polyacrylamide gels, protein bands from “starved” cells remained discrete, and despite low levels of incorporation, radiolabeled bands were detectable, suggesting that low levels of protein synthesis were sustained. A prominent band that accumulated in serum-starved cells was digested with trypsin and analyzed by tandem mass spectrometry, which identified the protein as eukaryotic elongation factor (EF)-1α EF-1α is well-conserved among species, and differential accumulation of EF-1α in serum-starved cells was verified by western blotting using a primary antibody to the homologous protein from Trypanosoma brucei. Aside from its importance in the elongation step of protein synthesis, EF-1α has been shown to have a number of non-canonical functions, including interaction with viral RNA and a potential role in apoptosis. We anticipate that the prolonged viability of mosquito cells in serum-free medium may provide a system to explore whether EF-1α accumulation is an adaptive response compatible with resumption of growth in the event that nutrients are replenished, or whether the excess EF-1α represents an irreversible commitment to an apoptotic pathway.

Quality Control of the Elongation Step of Protein Synthesis by tmRNP

Trans-translation is a quality-control process, activated upon premature termination of protein elongation, which recycles stalled ribosomes and degrades incomplete polypeptides. These functions are facilitated by transfer-messenger RNA (tmRNA, also called 10Sa RNA or SsrA RNA), a small stable RNA molecule encoded by the SsrA gene found in bacteria, chloroplasts and mitochondria. Most tmRNAs consist of a tRNA- and an mRNA-like domain connected by up to four pseudoknots. Comparative sequence analysis provided the first insight into tmRNA secondary and three-dimensional structure. Studies of the E. coli tmRNA in vitro and in vivo demonstrated that tmRNA functions as a ribonucleoprotein (RNP) complex with elongation factor Tu (EF-Tu), protein SmpB and ribosomal protein S1. The tRNA-like and mRNA-like activities of tmRNA mark prematurely terminated proteins for degradation by attaching to their C-termini peptide tags, which are recognized by numerous proteases. Studies aimed at understanding the details of the molecular mechanisms of trans-translation are ongoing.

Inhibition of protein synthesis in cortical neurons during exposure to hydrogen peroxide.

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate and zinc, as well as H(2)O(2) formation during the reperfusion period, strongly depresses protein synthesis. We have previously demonstrated that the glutamate-induced increase in cytosolic Ca(2+) is likely responsible for blockade of the elongation step of protein synthesis, whereas Zn(2+) preferentially inhibits the initiation step. In this study, we provide evidence indicating that H(2)O(2) and thapsigargin mobilized a common intracellular Ca(2+) pool. H(2)O(2) treatment stimulated a slow increase in intracellular Ca(2+), and precluded the effect of thapsigargin on Ca(2+) mobilization. H(2)O(2) stimulated the phosphorylation of both eIF-2alpha and eEF-2, in a time- and dose-dependent manner, suggesting that both the blockade of the elongation and of the initiation step are responsible for the H(2)O(2)-induced inhibition of protein synthesis. However, kinetic data indicated that, at least during the first 15 min of H(2)O(2) treatment, the inhibition of protein synthesis resulted mainly from the phosphorylation of eEF-2. In conclusion, H(2)O(2) inhibits protein translation in cortical neurons by a process that involves the phosphorylation of both eIF-2alpha and eEF-2 and the relative contribution of these two events depends on the duration of H(2)O(2) treatment.

Plasmodium protein phosphatase 2C dephosphorylates translation elongation factor 1beta and inhibits its PKC-mediated nucleotide exchange activity in vitro.

The elongation step of protein synthesis involves binding of aminoacyl-tRNA to the ribosomal A site, formation of a peptide bond and translocation of the newly formed peptidyl-tRNA to the P site. The nucleotide exchange factor EF-1beta plays a major role in the regulation of this process by regenerating a GTP-bound EF-1alpha necessary for each elongation cycle. EF-1beta has been shown to be phosphorylated and its phosphorylation is critical for optimal activity. We have previously identified a serine/threonine protein phosphatase 2C (PP2C) from the human malaria parasite Plasmodium falciparum. In the current work, we performed Far-Western analysis to identify PfPP2C substrates. Several components of the translation and transcription machinery were identified, including translation elongation factor 1-beta (PfEF-1beta). PfEF-1beta is efficiently phosphorylated by protein kinase C and this phosphorylation results in a 400% increase in its nucleotide exchange activity. PKC-phosphorylated PfEF-1beta is readily and selectively dephosphorylated by recombinant and native PfPP2C, which downregulates the nucleotide exchange activity to its basal level. The identification of a translation elongation component as substrate for PP2C suggests an important regulatory function for this enzyme and suggests that it may be a good target for drug design in the fight against malaria.

Molecular cloning of the gene encoding an acidic ribosomal protein of the P2 family from the ciliate Euplotes raikovi.

We have characterized a macronuclear gene of the ciliate protozoan Euplotes raikovi, which encodes an acidic ribosomal protein of the P protein family. This gene shows the typical organization of the hypotrich ciliate macronuclear "gene-sized" molecules with Euplotes telomeres at the ends. The longest open reading frame encodes a conceptual protein of 113 amino acid residues, with a molecular mass and pI value of 11.45 kDa and 3.97, respectively. By using sequence homology analysis, the protein was found to belong to the ribosomal P2 protein family and was named Er P2, where Er stands for Euplotes raikovi. These proteins, generally called A (acidic/alanine rich) proteins in prokaryotes and P (phosphorylated) proteins in eukaryotes, in which they are divided into P1 and P2 families, play a role in the elongation step of protein synthesis. Approximately 40% amino acid sequence identity was found between the cloned protein and other known protozoan ribosomal P2 proteins. Within its N-terminal half, this protein contains several potential kinase phosphorylation sites. Protein Er P2 differs markedly from the consensus P protein sequence in its C-terminal region, usually highly conserved among eukaryotic ribosomal P proteins, and shows similarities with the C-terminus of the archaebacterial ribosomal A proteins. To our knowledge, this E. raikovi protein represents the first demonstration of a ribosome-associated protein of the P2 family in a ciliate protozoan.

Genetic interactions between a null allele of the RIT1 gene encoding an initiator tRNA-specific modification enzyme and genes encoding translation factors in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase that exclusively modifies the initiator tRNA (tRNAMet(i)) by the addition of a 2'-O-ribosyl phosphate group to Adenosine 64. As a result, tRNAMet(i) is prevented from participating in the elongation steps of protein synthesis. We previously showed that the modification is not essential for the function of tRNAMet(i) in the initiation of translation, since rit1 null strains are viable and show no obvious growth defects. Here, we demonstrate that yeast strains in which a rit1 null allele is combined with mutations in any of the genes for the three subunits of eukaryotic initiation factor-2 (eIF-2), or with disruption alleles of two of the four initiator methionine tRNA (IMT) genes, show synergistic growth defects. A multicopy plasmid carrying an IMT gene can alleviate these defects. On the other hand, introduction of a high-copy-number plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor 1alpha (eEF-1alpha), into rit1 null strains with two intact IMT genes had the opposite effect, indicating that increased levels of eEF-1alpha are deleterious to these strains, presumably due to sequestration of the unmodified met-tRNAMet(i) for elongation. Thus, under conditions in which the components of the ternary met-tRNAMet(i):GTP:eIF-2 complex become limiting or are functionally impaired, the presence of the 2'-O-ribosyl phosphate modification in tRNAMet(i) is important for the provision of adequate amounts of tRNAMet(i) for formation of this ternary complex.

Nucleotide Sequence of a cDNA for a P2 60S Acidic Ribosomal Protein from Parthenium argentatum

In screening a stem-bark cDNA library from guayule (Parthenium argentatum) for genes involved in isoprenoid biogenesis, we isolated a full-length gene for a P2 60S acidic ribosomal protein that shows sequence similarity to vertebrate (Rich and Steitz, 1987), invertebrate (Qian et al., 1987), and yeast (Beltrame and Bianchi, 1987, 1990; Remacha et al., 1988) 60S acidic ribosomal proteins (Table I). The guayule P2 sequence is a full-length cDNA; a partial cDNA has been described for Arabidopsis (C. Bardet, M. Axelos, D. Tremousaygue, M. Lebas, T. Lagravere, and B. Lescure, unpublished data; EMBL accession number Z17464) with 65.5% identity at the nucleic acid level and 54.5% identity and 74.5% similarity at the amino acid level. Our interest in this gene extends to its potential use in experimental studies of Parthenium, from which few genes are at present available. Being a ribosomal protein, it should display minimal variations in expression and thus serve as a suitable internal control for gene expression studies in this species. The P2 60S acidic ribosomal protein plays a crucial role in the elongation step of protein synthesis. The P2 proteins exist as heterodimers composed of a and d subunits. Comparison of the P2 Parthenium sequence with both the a (Beltrame and Bianchi, 1987) and p (Beltrame and Bianchi, 1990) subunits of fission yeast shows nearly the same identity, 52.7 versus 50.9%, respectively, making it difficult to classify the guayule protein as P2 a or P2 F. Comparison of the P2 Parthenium sequence to the human LA2 protein (Rich and Steitz, 1987) shows 59.0% identity at the nucleic acid level and 54.5% identity and 79.5% similarity on the amino acid level, with a similar level of identity for the Drosophila gene (Qian et al., 1987).

Elongation factor EF-1 delta, a new target for maturation-promoting factor in Xenopus oocytes.

A new physiological target for Cdc2 protein kinase has been identified. It corresponds to a protein EF-1 delta, a constituent of the nucleotide exchange factor EF-1 beta gamma delta, involved in the elongation step of protein synthesis. EF-1 delta is phosphorylated by Cdc2 kinase on threonine and serine residues. Threonine has been identified as Thr122 in the sequence VQVTPAAK. During oocyte maturation, Thr122 is phosphorylated at metaphase, when p34cdc2 is active. Phosphorylation studies revealed the presence of two post-translational regulated forms of EF-1 delta protein. Identification of two isoforms of the delta protein, together with the presence of two guanine-nucleotide exchange proteins (beta and delta) and physiologically regulated phosphorylation sites by Cdc2 kinase on gamma and delta proteins, implicate that EF-1 beta gamma delta exists in the cell under a multitude of macromolecular forms which suggests that EF-1 beta gamma delta is a sophisticated regulatory factor rather than a "housekeeping" element of the cell.

Mechanism of action of purpuromycin.

Purpuromycin, an antibiotic active against both fungi and bacteria, shows different modes of action against these two kinds of micro-organisms; in Candida albicans it inhibits RNA synthesis, whereas in Bacillus subtilis protein synthesis is primarily affected, with DNA and RNA synthesis blocked at higher concentrations of the drug. In bacterial cell-free protein-synthesis systems, purpuromycin did not inhibit synthesis from endogenous mRNA (elongation of peptides initiated within the intact cell) but inhibited MS2-phase RNA-dependent protein synthesis (which requires initiation) by 50% at 0.1 mg/l. Poly(U)-directed polyphenylalanine synthesis was 50% inhibited by 20 mg of purpuromycin/l when added to a complete system; however, when purpuromycin was preincubated with ribosomes dissociated into 30 S and 50 S subunits, the concentration for 50% inhibition fell to 0.1 mg/l. By contrast, in a C. albicans cell-free system poly(U)-directed polyphenylalanine synthesis was partially inhibited only at 200 mg/l. Purpuromycin also inhibited polynucleotide synthesis in vitro in reactions using Escherichia coli or wheat-germ RNA polymerases or E. coli DNA polymerase I. We suggest that in bacteria the primary target of purpuromycin is on ribosomes and that its action precedes the elongation step of protein synthesis. The effect on nucleic acid synthesis in both fungi and bacteria may be due to interaction of purpuromycin with DNA.

Mutants of initiator tRNA that function both as initiators and elongators.

We describe the effect of mutations in the acceptor stem of Escherichia coli initiator tRNA on its function in vivo. The acceptor stem mutations were coupled to mutations in the anticodon sequence from CAU----CUA to allow functional studies on the mutant tRNAs in initiation and in elongation in vivo. We show that, with one exception, there is a good correlation between the kinetic parameters for formylation of the mutant tRNAs in vitro (preceding paper, Lee, C.P., Seong, B. L., and RajBhandary, U.L. (1991) J. Biol. Chem. 266, 18012-18017) and their activity in initiation in vivo. These results suggest an important role for formylation of initiator tRNA in its function in initiation, at least when it is aminoacylated with glutamine as is the case with the mutant tRNAs used here. Mutant tRNAs that have a base pair between nucleotides 1 and 72 at the top of the acceptor stem function as elongators, as analyzed by their ability to suppress an amber mutation in the E. coli beta-galactosidase gene. One of these mutants is also quite active in initiation. Thus, activities of a tRNA in initiation and elongation steps of protein synthesis are not mutually exclusive. Using a mRNA with two in frame UAG codons, we show that this mutant tRNA can both initiate protein synthesis from the upstream UAG and suppress the down-stream UAG. We discuss the potential use of tRNAs with such "dual" functions in tightly regulated expression of genes for proteins in E. coli.

Monoclonal antibodies against acidic phosphoproteins P0, P1, and P2 of eukaryotic ribosomes as functional probes.

Mice were immunized against ribosomal acidic proteins P1 and P2 from Artemia salina, and three kinds of monoclonal antibodies were isolated. One recognized P0 in addition to both P1 and P2 (anti-P). The other two recognized either P1 (anti-P1) or P2 (anti-P2) specifically and did not recognize P0. The anti-P antibody, but not anti-P1 or anti-P2, recognized a 22-amino acid peptide corresponding to the carboxyl-terminal sequence common to P1 and P2. This antibody, but not the others, inhibited poly(U)-directed polyphenylalanine synthesis. The anti-P1 bound to ribosomes but failed to inhibit polyphenylalanine synthesis: the anti-P2 did not bind to ribosomes at all. The anti-P and its Fab fragments inhibited the elongation step of protein synthesis, namely, the binding of elongation factors 1 alpha and 2 to ribosomes as well as their ribosome-coupled GTPase activities. Anti-P had little effect on the nonenzymatic phenylalanyl-tRNA binding to ribosomes and on peptidyltransferase activity. These results suggest the functional importance of the homologous carboxyl-terminal region of the three P proteins for the interaction of the ribosome with the two elongation factors. The epitope of anti-P1 must reside in a region of the protein which is not directly involved in its function.

Isolation and characterization of theDrosophilatranslational elongation factor 2 gene

We have isolated a cDNA clone that encodes the Drosophila melanogaster elongation factor 2 (EF2), a protein involved in the elongation step of protein synthesis. This identification was based on the high degree of its amino acid sequence identity (greater than 80%) to that of hamster EF2. The gene encoding Drosophila EF2 is found at position 39E-F of the 2L chromosomal arm and maybe identical to the M(2)H locus, which produces a Minute phenotype when mutated. The genomic organization of the locus includes four exons. Conserved sequence segments shared with a variety of GTP binding proteins are found in the amino terminal third of the protein, and segments unique to EF2 and its prokaryotic functional homolog, EF-G, are in the carboxy terminal half; these two regions are segregated in two respective exons.

Role of Protein Synthesis in Decay and Accumulation of mRNA during Spore Germination in the Cellular Slime Mold Dictyostelium discoideum

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific mRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis inhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of inhibitors, albeit at a diminished rate relative to that of the uninhibited control.

Identification of ribosomal protein autoantigens.

Approximately 20% of patients with systemic lupus erythematosus and with anti-Sm autoantibodies synthesize autoantibodies, called anti-rRNP, to components of the ribosome. We found that anti-rRNP sera reacted predominantly with three ribosomal phosphoproteins of approximate Mr = 38,000, 16,000 and 15,000, both by immunoprecipitation and by immunoblotting. The human autoantibodies cross-reacted with similar antigens present in rodent, brine shrimp, and yeast cells but reacted weakly if at all with proteins of bacteria. Thus the human autoantibodies recognize epitopes that are widely conserved in evolution. Purified ribosomal proteins together with specific rabbit antisera were used to identify the two smaller rRNP antigens as the acidic phosphoproteins of the large ribosomal subunit, designated P1/P2(L40/L41) (rat), eL7/eL12 (Artemia, brine shrimp), and A1/A2 (yeast). These proteins function in the elongation step of protein synthesis in an analogous fashion to the L7/L12 ribosomal proteins of E. coli. The 38,000-dalton rRNP antigen corresponds to a nonacidic protein also associated with the large ribosomal subunit. The human autoantibodies appear to have a specificity similar to that of a previously described mouse monoclonal antibody obtained from mice injected with heterologous (chick) ribosomes, suggesting that both the human polyclonal autoantibodies and the mouse monoclonal recognize a class of epitope(s) that is common in all three ribosomal proteins. In addition, we found that many of the anti-ribosomal sera contained a further class of autoantibodies reactive with naked RNA. These may be similar to the anti-RNA antibodies previously described in both humans and mice with autoimmune disease.

Queuosine modification of the wobble base in tRNAHis influences 'in vivo' decoding properties.

The 'in vivo' decoding properties of four tRNAHis isoacceptors, two from Drosophila melanogaster and two from brewer's yeast, were studied after their microinjection, along with turnip yellow mosaic virus (TYMV) coat protein mRNA, into Xenopus laevis oocytes. The two Drosophila isoacceptors are identical besides containing either a guanosine (G) or the hypermodified nucleoside queuosine (Q) in the wobble position. The brewer's yeast isoacceptors differ by four bases in the anticodon stem, and by one base in the amino acceptor stem. Our results show that, under competing 'in vivo' conditions, the Drosophila tRNAHis with the anticodon GUG clearly prefers the histidine codon CAC to the codon CAU, whereas little preference is observed for the tRNAHis with the anticodon QUG for the codon CAU, and no preference for either codon by the two yeast isoacceptors. Hence, it can be concluded that the presence of the Q-base clearly affects the choice of the codon. This is the first demonstration of an 'in vivo' codon preference by tRNA isoacceptors differing in the modification of the wobble base during the elongation step of protein synthesis. These results imply that one function of the Q-base is at the translational level.