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Inferring rules of Escherichia coli translational efficiency using an artificial neural network.

Although the machinery for translation initiation in Escherichia coli is very complicated, the translational efficiency has been reported to be predictable from upstream oligonucleotide sequences. Conventional models have difficulties in their generalization ability and prediction nonlinearity and in their ability to deal with a variety of input attributions. To address these issues, we employed structural learning by artificial neural networks to infer general rules for translational efficiency. The correlation between translational activities measured by biological experiments and those predicted by our method in the test data was significant (r=0.78), and our method uncovered underlying rules of translational activities and sequence patterns from the obtained skeleton structure. The significant rules for predicting translational efficiency were (1) G- and A-rich oligonucleotide sequences, resembling the Shine-Dalgarno sequence, at positions -10 to -7; (2) first base A in the initiation codon; (3) transport/binding or amino acid metabolism gene function; (4) high binding energy between mRNA and 16S rRNA at positions -15 to -5. An additional inferred novel rule was that C at position -1 increases translational efficiency. When our model was applied to the entire genomic sequence of E. coli, translational activities of genes for metabolism and translational were significantly high.

Base Sequence↗

A somatic mutation in the 5'UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency.

Mutations in the 5' UTR which cause increment/decrement of translation efficiency have been recently described as a novel molecular mechanism of disease. Alterations in the consensus sequence for the translation initiation may promote context-dependent leaky scanning of ribosomes and/or initiation from a downstream AUG codon. Initiation of translation from a downstream in-frame AUG codon in BRCA1 gene was recently identified in normal cells and possibly in breast cancer. Here we present further insight into BRCA1 translational pathophysiology investigating the role of the canonical structure of the initiation consensus sequence of BRCA1. We have analysed the effect of a somatic point mutation (117 G>C) in position -3 with respect to the AUG of the BRCA1 gene, identified in a highly aggressive sporadic breast cancer. We constructed chimeric genes encoding the luciferase reporter sequence downstream of the wild type or the mutated BRCA1 5'UTR. These transcripts were tested for their activity in in vitro and in vivo systems. In in vitro transcription/translation assays the estimated translation efficiency of the construct with the mutated BRCA1 5'UTR was 30-50% lower than that with the wild type BRCA1 5'UTR. The same chimeric genes were analysed for their expression in vivo by transient transfection in human cells. While the two constructs were equally transcribed, the plasmid carrying the mutated sequence produced 70% less luciferase activity compared to the wild type sequence. Finally, to obtain a direct evaluation on translational efficiency in vivo, we analysed mRNA translation on translationally active and non-active ribosomes separated from transfected cells. Mutant mRNA was partially localized in subpolysomal particles analytically confirming a polysome recruitment defect. Thus, characterization of BRCA1 5'UTR and translation efficiency seems to provide new insight into BRCA1 role in breast and ovarian cancer pathogenesis.

5' Untranslated Regions↗

Influence of translational efficiency on the stability of the mRNA for ribosomal protein S20 in Escherichia coli.

A set of plasmids was constructed so as to contain point mutations which limit the efficiency and/or extent of translation of the gene for ribosomal protein S20. These plasmids were transformed into strains carrying mutations in the genes for polynucleotide phosphorylase (pnp-7), RNase E (rne-1), or both. Subsequently, the effect of translational efficiency on mRNA abundance and chemical half-life was determined. The data indicated that mutations altering translational efficiency also affected mRNA levels over a 10-fold range. This variation in mRNA abundance occurred independently of mutations in either RNase E or polynucleotide phosphorylase, both of which determine the stability of the S20 mRNAs. Moreover, a mutation at codon 15 which caused premature termination of translation of the S20 mRNA did not significantly reduce its stability in different genetic backgrounds. We propose a model in which initiation of translation competes for early steps in mRNA turnover, which could be the binding of RNase E itself or as a complex to one or more sites near the 5' terminus of the S20 mRNA.

Base Sequence↗

Parameters influencing translational efficiency in aphthovirus IRES-based bicistronic expression vectors.

Initiation of translation in picornavirus RNAs occurs internally, mediated by an internal ribosome entry site (IRES) element. This property has been exploited to coexpress proteins from a single bicistronic transcription unit in eukaryotic cells. The region that separates the IRES element from the authentic initiator codon of the second gene plays an important role in the translation efficiency of this cistron. In the present report, we have analyzed the effect of sequence modifications in this region on the translation efficiency directed by the foot-and-mouth disease (FMDV) IRES in bicistronic expression vectors. Insertion of various sequences, which contained additional start codons and/or the capacity to form hairpins immediately downstream of the 3' border of the IRES, strongly reduced the translation efficiency of the second gene in bicistronic RNAs. Interestingly, an increase of distance per se did not have a deleterious effect on translation efficiency. The bicistronic vector studied here tolerated 95 nucleotides between the 3' border of the IRES and the authentic start codon, provided that out-of-frame AUG codons or hairpins were not present in this RNA segment. These results indicate that FMDV-derived bicistronic constructs are extremely well suited for use in eukaryotic expression vectors.

Animals↗

Immunogene therapy of murine fibrosarcoma using IL-15 gene with high translation efficiency.

We have recently found that translational efficiency is up-regulated by an alternative exon in IL-15 mRNA in mice. In a malignancy model using BALB/c mice and syngeneic Meth A fibrosarcoma (Meth A), we successfully applied immunological gene therapy with IL-15 protein using alternative IL-15 cDNA with high translational efficiency. Two expression vectors carrying the murine IL-15 gene were constructed for use in tumor immunotherapy, one utilizing IL-15 cDNA with alternative exon 5 and the second utilizing IL-15 cDNA with normal exon 5. The first vector induced the production of a large amount of IL-15 protein in Meth A cells, whereas tumor cells transfected by the second vector produced only a marginal level of IL-15 protein. Although cell growth of both transfectants in vitro remained unchanged, inoculation of clones transfected with normal IL-15 cDNA resulted in progressive tumor growth, while clones transfected with alternative IL-15 cDNA led to the rejection of the tumor. The clone producing high levels of IL-15 grew progressively in nude mice and mice treated with anti-CD4 monoclonal antibodies (mAb), whereas the growth of the transfectants was retarded in anti-CD8 mAb- or anti-asialo GM1 antibody-treated mice. Cured mice were shown to have generated immunity against a subsequent challenge with the wild type of Meth A but not against Meth 1 tumor cells, another type of fibrosarcoma derived from BALB/c mice. Thus, tumor therapy based on IL-15 gene transfection was effective against Meth A tumor cells, suggesting a possible application to human neoplasms.

Animals↗

Effect of leader primary structure on the translational efficiency of phosphoglycerate kinase mRNA in yeast.

In order to determine the effect of the nucleotide composition of the 5'-untranslated (leader) region on the translational efficiency of mRNA in yeast, we replaced a large part of the leader region of the phosphoglycerate kinase (PGK) gene by various deoxyoligonucleotides of defined sequence. All mutations left the context of the transcription initiation site and AUG start codon intact. The mutant genes were introduced into yeast cells on a multicopy vector and the ratio of the steady-state levels of PGK mRNA and protein were determined. We found translational efficiency to be unaffected by the presence of either an 18 nucleotides (nt) long polyA or polyC tract or by sequences consisting of mixtures of A and C residues in any proportion. In contrast, a poly U tract, as well as mixtures of U and C residues, reduced translational efficiency by a factor of two to three, presumably by long-range base-pairing between the leader and sequences elsewhere in the coding or 3'-non-coding regions of the messenger. In agreement with this hypothesis, a five-fold reduction in translational efficiency was found for an mRNA carrying a polyC tract in the leader as well as a polyG tract in the trailer, neither of which had any effect on translational efficiency by itself. Therefore, we conclude that the leader and trailer regions (including the polyA tail) of PGK mRNA are sufficiently close to base-pair when containing complementary sequences. The resulting secondary structure evidently constitutes a barrier for incoming 40S subunits on their way to the AUG start codon. The presence of an 18 nt long polyG tract in the leader completely abolished translation of the PGK mRNA in accordance with earlier observations. However, we found that leaders containing up to 40% G residues interspersed with either A or U, still allow highly efficient translation. This value is about four times as high as the average G content of leader sequences in naturally occurring yeast mRNAs. Finally, neither deletion of about 40% of the trailer sequence of PGK mRNA, nor replacement of this sequence by homopolymer tracts had any effect on translational efficiency.

Amino Acid Sequence↗

Repetitive elements in the 5' untranslated region of a human zinc-finger gene modulate transcription and translation efficiency.

A substantial proportion of the human genome consists of repetitive sequences. Although most of these sequences are nonessential for the organism, retroelements, such as Alu sequences, L1s, and HERVs (human endogenous retroviruses), have recently been implicated in the regulation of various genes. Our laboratory previously identified a novel, alternatively spliced zinc-finger gene, ZNF177, which incorporates Alu L1, and HERV segments into the 5' untranslated region (UTR) of transcripts. In this study, we investigated the genomic structure and functional significance of the repetitive sequences in the 5' UTR of ZNF177 mRNAs. Using luciferase and GFP reporter constructs, we assessed the effect of the HERV, Alu, and L1 sequences on gene expression levels. Our results indicate that the presence of the retroelement sequences, particularly the Alu and L1 segments which form one 5' UTR exon, modifies the expression level of both reporter genes. We present evidence that the Alu and L1 sequences alter both the RNA and protein levels of reporter genes by increasing transcription efficiency while decreasing translation efficiency. Our findings indicate that the Alu and L1 repeats in the 5' UTR of ZNF177 exert a positive transcriptional enhancer effect, but repress translation of the zinc finger gene. In addition, our analysis of a 5' UTR database suggests that 4% of human 5' UTRs harbor Alu sequences, indicating that the expression of many genes might be influenced by Alu repeats. These results illustrate the complex regulatory effects that retroelements can have on human gene expression.

5' Untranslated Regions↗

Control of the translational efficiency of beta-F1-ATPase mRNA depends on the regulation of a protein that binds the 3' untranslated region of the mRNA.

The expression of the nucleus-encoded beta-F1-ATPase gene of oxidative phosphorylation is developmentally regulated in the liver at both the transcriptional and posttranscriptional levels. In this study we have analyzed the potential mechanisms that control the cytoplasmic expression of beta-F1-ATPase mRNA during liver development. Remarkably, a full-length 3' untranslated region (UTR) of the transcript is required for its efficient in vitro translation. When the 3' UTR of beta-F1-ATPase mRNA is placed downstream of a reporter construct, it functions as a translational enhancer. In vitro translation experiments with full-length beta-F1-ATPase mRNA and with a chimeric reporter construct containing the 3' UTR of beta-F1-ATPase mRNA suggested the existence of an inhibitor of beta-F1-ATPase mRNA translation in the fetal liver. Electrophoretic mobility shift assays and UV cross-linking experiments allowed the identification of an acutely regulated protein (3'betaFBP) of the liver that binds at the 3' UTR of beta-F1-ATPase mRNA. The developmental profile of 3'betaFBP parallels the reported changes in the translational efficiency of beta-F1-ATPase mRNA during development. Fractionation of fetal liver extracts revealed that the inhibitory activity of beta-F1-ATPase mRNA translation cofractionates with 3'-UTR band-shifting activity. Compared to other tissues of the adult rat, kidney and spleen extracts showed very high expression levels of 3'betaFBP. Translation of beta-F1-ATPase mRNA in the presence of kidney and spleen extracts further supported a translational inhibitory role for 3'betaFBP. Mapping experiments and a deletion mutant of the 3' UTR revealed that the cis-acting element for binding 3'betaFBP is located within a highly conserved region of the 3' UTR of mammalian beta-F1-ATPase mRNAs. Overall, we have identified a mechanism of translational control that regulates the rapid postnatal differentiation of liver mitochondria.

ADP-Ribosylation Factors↗

Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts.

Codon usage in chloroplasts is different from that in prokaryotic and eukaryotic nuclear genomes. However, no experimental approach has been made to analyse the translation efficiency of individual codons in chloroplasts. We devised an in vitro assay for translation efficiencies using synthetic mRNAs, and measured the translation efficiencies of five synonymous codon groups in tobacco chloroplasts. Among four alanine codons (GCN, where N is U, C, A or G), GCU was the most efficient for translation, whereas the chloroplast genome lacks tRNA genes corresponding to GCU. Phenylalanine and tyrosine are each encoded by two codons (UUU/C and UAU/C, respectively). Phenylalanine UUC and tyrosine UAC were translated more than twice as efficiently than UUU and UAU, respectively, contrary to their codon usage, whereas translation efficiencies of synonymous codons for alanine, aspartic acid and asparagine were parallel to their codon usage. These observations indicate that translation efficiencies of individual codons are not always correlated with codon usage in vitro in chloroplasts. This raises an important issue for foreign gene expression in chloroplasts.

Amino Acid Sequence↗

Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region.

We test the hypothesis that the translation machinery in cells infected by influenza A virus efficiently translates only mRNAs that possess the influenza viral 5' untranslated region (5'-UTR) by introducing mRNAs directly into the cytoplasm of infected cells. This strategy avoids effects due to the inhibition of the nuclear export of cellular mRNAs mediated by the viral NS1 protein. In one approach, we transfect in vitro synthesized mRNAs into infected cells and demonstrate that these mRNAs are efficiently translated whether or not they possess the influenza viral 5'-UTR. In the second approach, an mRNA is synthesized endogenously in the cytoplasm of influenza A virus infected cells by a constitutively expressed T7 RNA polymerase. Although this mRNA is uncapped and lacks the influenza viral 5'-UTR sequence, it is efficiently translated in infected cells via an internal ribosome entry site. We conclude that the translation machinery in influenza A virus infected cells is capable of efficiently translating all mRNAs and that the switch from cellular to virus-specific protein synthesis that occurs during infection results from other processes.

5' Untranslated Regions↗

Structural features of myelin basic protein mRNAs influence their translational efficiencies.

The myelin basic protein (MBP) gene expresses several alternatively spliced products with the same 5' and 3' untranslated regions (UTRs). It has been reported that its expression may be regulated not only at the transcriptional level but also at the translational level during development. We engineered several MBP mRNA deletion mutants with 5' (-48, -37, -27, -22, and -10) and 3' UTRs of differing lengths and examined the translational efficiencies of these constructs in cell-free systems. The translational efficiencies of the constructs differed significantly over a range of almost 10-fold. A deletion of 11 nucleotides from the 5' end of the natural (i.e., -48) MBP mRNA resulted in an approximate fourfold reduction in translational efficiency. Further truncation of the 5' UTR increased the translational efficiencies of the constructs as has been observed with many RNAs. These results suggest that there may be a positive control element between -48 and -37 nucleotides in the 5' UTR of MBP mRNA. The effects of modifying the lengths of the 5' UTR on the translational efficiency of mRNAs encoding the 21.5-kDa and 14-kDa MBPs were the same, suggesting that the effect observed was not unique to the 21.5-kDa MBP mRNA. Truncating the 3' UTR of four different alternatively spliced MBP mRNAs also altered their translational efficiencies. Thus, the 5' and 3' UTRs of MBP mRNAs appear to influence the translation of these mRNAs, and such factors may be involved in the translational regulation of MBP gene expression.

Animals↗

Identification of two critical base pairings in 5' untranslated regions affecting translation efficiency of synthetic uncapped globin mRNAs.

We observed a marked difference between the in vitro translation efficiency of two uncapped synthetic mRNAs, displaying the entire human alpha or beta globin mRNA sequences and some additional non-globin sequences in 5'. The comparison of the translation efficiencies of chimeric mRNAs indicated that the alpha 5' untranslated region (5' UTR) is responsible for a low translation efficiency that cannot be explained neither by primary sequence nor by the overall stability of 5' UTR secondary structures only. By point mutations in this alpha 5' UTR, we identified two base pairings at position -1 and -2 preceding the initiation codon which are associated with a negative effect on translation efficiency.

Base Composition↗

Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis.

We have quantitatively analyzed the relationship between translational efficiency and the mRNA secondary structure in the initiation region. The stability of a defined hairpin structure containing a ribosome binding site was varied over 12 kcal/mol (1 cal = 4.184 J) by site-directed mutagenesis and the effects on protein yields were analyzed in vivo. The results reveal a strict correlation between translational efficiency and the stability of the helix. An increase in its delta G0 of -1.4 kcal/mol (i.e., less than the difference between an A.U and a G.C pair) corresponds to the reduction by a factor of 10 in initiation rate. Accordingly, a single nucleotide substitution led to the decrease by a factor of 500 in expression because it turned a mismatch in the helix into a match. We find no evidence that exposure of only the Shine-Dalgarno region or the start codon preferentially favors recognition. Translational efficiency is strictly correlated with the fraction of mRNA molecules in which the ribosome binding site is unfolded, indicating that initiation is completely dependent on spontaneous unfolding of the entire initiation region. Ribosomes appear not to recognize nucleotides outside the Shine-Dalgarno region and the initiation codon.

Base Sequence↗

The 5' and 3' untranslated regions of satellite tobacco necrosis virus RNA affect translational efficiency and dependence on a 5' cap structure.

Satellite tobacco necrosis virus RNA (STNV RNA) is a naturally uncapped viral RNA that contains 1239 nucleotides: 29 in the 5' untranslated region (UTR), 591 in the coding region and 619 in the 3' UTR. Mutations were made in the 5' and 3' UTRs, and the effects of these mutations on translational efficiency and cap independence were measured in an in vitro translation system from wheat germ. Removal of the first 12 nucleotides or 10 changes in the nucleotide sequence of the 5' UTR reduced translational efficiency approximately 3-fold; capping of these 5' mutant mRNAs restored their translational efficiencies. Truncation of the 3' UTR to nucleotide 627 or 700, or deletion of nucleotides 627-737, reduced translational efficiency more than 20-fold; capping of these 3' mutant mRNAs restored their translational efficiencies. These modifications in the 3' UTR increased the concentration of initiation factor 4F required for translation. Chimeric mRNAs were constructed which contained the coding region of rabbit alpha-globin mRNA and either the 5' UTR, 3' UTR, or both the 5' and 3' UTRs of STNV RNA. Both the 5' and 3' UTRs of STNV RNA were necessary to obtain cap-independent translation. These findings indicate that interaction between 5' UTR and the region between nucleotides 627 and 737 in the 3' UTR are required for cap-independent translation.

Animals↗

Correlation between poly(U) misreading and poly(dT) translation efficiency in E coli cell-free systems.

A positive correlation between poly(U) misreading and efficiency of poly(dT) translation has been revealed in cell-free systems from wild-type E coli and streptomycin--resistant mutants with altered ribosomal protein S12. Different factors promoting misreading of poly(U) such as aminoglycoside antibiotics and Mg2+ ions also stimulate poly(dT) translation. The effect of the antibiotics on poly(U) translation efficiency and misreading as well as on poly(dT) decoding is characterised by the same order: neomycin greater than kanamycin greater than streptomycin. S12 mutants ribosomes are less erroneous in poly(U) translation and less efficient in poly(dT) decoding. The data obtained are in good agreement with the hypothesis of stereospecific stabilization of codon-anticodon complexes by the ribosome decoding centre.

Anti-Bacterial Agents↗

Translational regulation of mitochondrial differentiation in neonatal rat liver. Specific increase in the translational efficiency of the nuclear-encoded mitochondrial beta-F1-ATPase mRNA.

Postnatal (1-h) mitochondrial differentiation in normal neonatal rat liver is regulated at the translational level (Izquierdo, J. M., Luis, A. M., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 9090-9097). The rapid postnatal increase in liver global rate of protein synthesis preferentially affects mitochondrial proteins (Valcarce, C., Navarrete, R. M., Encabo, P., Loeches, E., Satrústegui, J., and Cuezva, J. M. (1988) J. Biol. Chem. 263, 7767-7775). Analysis of polysome profiles and determination of both eukaryotic initiation factor 2 (eIF-2) activity and amount of eIF-2 beta protein in the liver of fetal and 1-h-old neonatal rats, indicate a rapid activation of translation initiation without changes in the amount of the translational machinery available between both stages of liver development. Appearance of a more acidic eIF-2 beta-subunit form in two-dimensional Western blots from 1-h-old rat livers suggests that covalently regulated modifications of the initiation factor phosphoproteins might be responsible for increased translation in the neonatal liver. On the other hand, preferential cytosolic translation of the mitochondrial nuclear-encoded beta-F1-ATPase mRNA at this stage of liver development is accomplished by (i) the antenatal accumulation of this mRNA in the fetal liver in 5-6-fold excess than that found in adults, although fetal liver beta-F1-ATPase mRNA shows negligible translational efficiency when compared to the adult counterpart; (ii) a 2-fold increase of the stored beta-F1-ATPase mRNA being rapidly mobilized into cytosolic polyribosomes, and (iii) a 3-fold increase in the in vitro determined translational efficiency of beta-F1-ATPase mRNA. Increased translational efficiency of beta-F1-ATPase mRNA at 1-h postnatal is specific for the nuclear-encoded template since beta-tubulin mRNA did not show any postnatal alteration in its translational efficiency. The results presented suggest that developmental changes in the poly(A)+ RNA fraction or in the reporter template itself are responsible for the increased and preferential translation of the nuclear-encoded mitochondrial mRNAs needed for mitochondrial differentiation and, thus, for mammalian adaptation to the extrauterine environment.

Animals↗

Translational efficiency of cMyc mRNA in Burkitt lymphoma cells.

The translational efficiency of cMyc mRNAs was assessed in a variety of cell lines: HeLa cells and Epstein-Barr virus-transformed lymphocytes, both of which contain only the germ line cMyc allele; Daudi, a Burkitt cell line containing a translocated cMyc gene with no apparent alteration; and P3HR-1, a Burkitt line in which the 5' end of the translocated cMyc gene has been altered by the chromosomal translocation. Translational efficiency was inferred by measuring the number of ribosomes associated with the cMyc mRNA, using a procedure by which individual polysomal fractions were analyzed by blot hybridization. Since polysome size is a function of the length of the translated sequence as well as the rate of initiation of protein synthesis, we also determined the number of ribosomes associated with a control mRNA (alpha tubulin) which codes for a protein of similar size to cMyc. We found that the cMyc mRNA was associated with a number of ribosomes comparable to that associated with alpha tubulin mRNA in all the cell lines tested.

Alleles↗

Efficient translation of the UAG termination codon in Candida species.

Clinical isolates of the dimorphic fungus Candida albicans encode a tRNA that, in a cell-free translation system prepared from the yeast Saccharomyces cerevisiae, efficiently translates the amber (UAG) termination codon. Unusually, the efficiency of this UAG read-through in the heterologous cell-free system is not further enhanced by polyamines. The suppressor tRNA is also able to efficiently translate the UAG codon in the rabbit reticulocyte cell-free system and with efficiencies approaching 100% in a homologous (C. albicans) cell-free system. That the suppressor tRNA is nuclear-encoded is demonstrated by the lack of activity in purified C. albicans mitochondrial tRNAs. Finally, UAG suppressor tRNA activity is also demonstrated in three other pathogenic Candida species, C. parapsilosis, C. guillermondii and C. tropicalis. These results suggest that some, but not all, Candida species have evolved an unusual nuclear genetic code in which UAG is used as a sense codon.

Candida albicans↗