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PubMed2006

Correlation of mRNA expression and protein abundance affected by multiple sequence features related to translational efficiency in Desulfovibrio vulgaris: a quantitative analysis.

The modest correlation between mRNA expression and protein abundance in large-scale data sets is explained in part by experimental challenges, such as technological limitations, and in part by fundamental biological factors in the transcription and translation processes. Among various factors affecting the mRNA-protein correlation, the roles of biological factors related to translation are poorly understood. In this study, using experimental mRNA expression and protein abundance data collected from Desulfovibrio vulgaris by DNA microarray and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) proteomic analysis, we quantitatively examined the effects of several translational-efficiency-related sequence features on mRNA-protein correlation. Three classes of sequence features were investigated according to different translational stages: (i) initiation, Shine-Dalgarno sequences, start codon identity, and start codon context; (ii) elongation, codon usage and amino acid usage; and (iii) termination, stop codon identity and stop codon context. Surprisingly, although it is widely accepted that translation initiation is the rate-limiting step for translation, our results showed that the mRNA-protein correlation was affected the most by the features at elongation stages, i.e., codon usage and amino acid composition (5.3-15.7% and 5.8-11.9% of the total variation of mRNA-protein correlation, respectively), followed by stop codon context and the Shine-Dalgarno sequence (3.7-5.1% and 1.9-3.8%, respectively). Taken together, all sequence features contributed to 15.2-26.2% of the total variation of mRNA-protein correlation. This study provides the first comprehensive quantitative analysis of the mRNA-protein correlation in bacterial D. vulgaris and adds new insights into the relative importance of various sequence features in prokaryotic protein translation.

Bacterial Proteins↗

PubMed2003

5' Untranslated region of the HSP18.2 gene contributes to efficient translation in plant cells.

Maximizing the amount of protein translated per unit mRNA is an important goal in establishing expression systems. The 5' untranslated region (5'-UTR) of mRNA is known to play an important role in determining the rate of translation. The full length 5'-UTR from the Arabidopsis thaliana heat shock protein (HSP) gene,HSPI8.2 gene, was inserted into the cloning site between the cauliflower mosaic virus 35S RNA promoter and beta-glucuronidase (GUS) gene in the pBI221 plasmid. When this construct was transfected into Nicotiana tabacum (tobacco) BY-2 protoplasts, the level of protein was about 10-fold higher than that of unmodified pB1221. The accumulation of each transcript was the same level. We also demonstrated that the 5'-UTR of the HSP18.2 gene enhances the rate of translation in stable transgenic BY-2 clones and Arabidopsis T87 protoplasts. The 5'-UTRs of the other Arabidopsis HSP genes -HSP17.4, HSP81-1,HSP81-2, andHSP81-3 - also conferred efficient translation. These 5'-UTRs ofHSP genes may be of use in increasing the expression of foreign proteins. In combination with a strong promoter, it can be used in the development of efficient protein production systems.

Journal Article↗

PubMed1987

Phosphorylation of ribosomal protein S6 during lens cell differentiation: correlation with translational efficiency.

In vitro differentiation of embryonic chicken lens epithelial explants to form lens fiber cells is accompanied by an increase in protein synthesis without a corresponding increase in mRNA levels. This apparent increase in translational efficiency is correlated with a specific enhancement of phosphorylation of a 32K protein, which we identify as ribosomal protein S6 by two dimensional gel electrophoresis of purified ribosomal proteins. Serum, insulin, and chicken vitreous humor, three agents known to initiate differentiation in this system, all lead to enhanced S6 phosphorylation. Maximal enhancement of phosphorylation is reached within the first hour after the onset of differentiation, and is not blocked by inhibitors of RNA and protein synthesis.

Animals↗

PubMed1992

Sequence requirements for efficient translational frameshifting in the Escherichia coli dnaX gene and the role of an unstable interaction between tRNA(Lys) and an AAG lysine codon.

Synthesis of the gamma-subunit of DNA polymerase III holoenzyme depends on precise and efficient translational frameshifting to the -1 frame at a specific site in the dnaX gene of Escherichia coli. In vitro mutagenesis of this frameshift site demonstrated the importance of an A AAA AAG heptanucleotide sequence, which allows two adjacent tRNAs to retain a stable interaction with mRNA after they slip to the -1 position. The AAG lysine codon present in the 3' half of this heptanucleotide was a key element for highly efficient frameshifting. A tRNA(Lys) with a CUU anticodon, which has a strong affinity for AAG lysine codons, is present in eukaryotic cells but absent in E. coli. Expression in E. coli of a mutant tRNA(Lys) with a CUU anticodon specifically inhibited the frameshifting at the AAG codon, suggesting that the absence of this tRNA in E. coli contributes to the efficiency of the dnaX frameshift.

Base Sequence↗

PubMed2004

Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency.

Synthetic analogs of the N7-methylated guanosine triphosphate cap at the 5' end of eukaryotic mRNAs and snRNAs have played an important role in understanding their splicing, intracellular transport, translation, and turnover. We report here a new series of N7-benzylated dinucleoside tetraphosphate analogs, b7Gp4G, b7m(3'-O)Gp4G, and b7m2Gp4G, that extend our knowledge of the role of the cap in translation. We used these novel analogs, along with 10 previously synthesized analogs, to explore five parameters: binding affinity to eIF4E, inhibition of cap-dependent translation in a rabbit reticulocyte lysate system, efficiency of incorporation into RNAs during in vitro transcription (% capping), orientation of the analog in the synthetic mRNA (% correct orientation), and in vitro translational efficiency of mRNAs capped with the analog. The 13 cap analogs differed in modifications of the first (distal) and second (proximal) guanine moieties, the first and second ribose moieties, and the number of phosphate residues. Among these were analogs of the naturally occurring cap m3(2,2,7)Gp3G. These compounds varied by 61-fold in affinity for eIF4E, 146-fold in inhibition of cap-dependent translation, 1.4-fold in % capping, and 5.6-fold in % correct orientation. The most stimulatory analog enhanced translation 44-fold compared with uncapped RNA. mRNAs capped with b7m2Gp4G, m7Gp3m7G, b7m(3'-OGp4G, and m7Gp4m7G were translated 2.5-, 2.6-, 2.8-, and 3.1-fold more efficiently than mRNAs capped with m7Gp3G, respectively. Relative translational efficiencies could generally be explained in terms of cap affinity for eIF4E, % capping, and % correct orientation. The measurement of all five parameters provides insight into factors that contribute to translational efficiency.

Animals↗

PubMed2004

Biomonitoring of Gulf of Patras, N. Peloponnesus, Greece. Application of a biomarker suite including evaluation of translation efficiency in Mytilus galloprovincialis cells.

Specimens of Mytilus galloprovincialis were placed in bow nets and immersed at 3-10 m depth in a clean coastal region (reference area), Itea, and two marine stations along Gulf of Patras, N. Peloponnesus, Greece. One site is near the estuaries of the Glafkos River, which are influenced by local industrial and urban sources (Station 1); the second site, Agios Vasilios, has no evident organic pollution but is enriched in metals, particularly zinc (Station 2). One month after immersion, digestive glands were removed from the mussels and tested for lysosomal membrane stability, metallothionein content, and translational efficiency of ribosomes. In addition, gill cells were isolated and their micronuclei content was determined. Compared with the reference samples, mussels transplanted to Gulf of Patras showed a significant increased lysosomal membrane permeability and metallothionein content, reduced polysome levels, and increased chromosomal damage in relation to the contamination burden of each sampling area. Also, runoff ribosomes from mussels transplanted to Gulf of Patras (that is, ribosomes stripped of endogenous messengers and peptidyl- or/and aminoacyl-tRNAs) were less efficient at initiating protein synthesis in an in vitro-translation system than those prepared from reference samples. The whole set of data suggests that the degree of Gulf of Patras pollution differs between different sites and depends on the proximity of urban sewage and industrial outfalls. In addition, our results emphasize the importance of protein synthesis regulation as a component of the cellular stress response.

Animals↗

PubMed1989

Effect of deletions in the 5'-noncoding region on the translational efficiency of phosphoglycerate kinase mRNA in yeast.

Deletions of various sizes were introduced into the region of the yeast PGK gene encoding the 5'-nontranslated portion of the phosphoglycerate kinase (PGK) mRNA. The effect of these deletions on the translational efficiency of the mutant transcripts was analysed by assaying the levels of mutant PGK mRNA and PGK protein in cells transformed with the mutant genes. Quantification of transcript levels by either Northern analysis or a reverse transcription assay demonstrated that there were no significant differences in the levels of mutant PGK mRNA between the various mutants. Thus, the leader sequence does not appear to play a role in determining the relatively long half-life of yeast PGK mRNA. Analysis of PGK protein levels in the various mutants revealed no effect when the length of the leader was reduced from 45 to 27 nucleotides (nt). Protein levels dropped by about a factor 2, however, upon a further decrease to 21 nt. Additional shortening did not cause a further dramatic reduction in translational yield. Even an mRNA containing a leader of only 7 nt was still translated at about 50% of the optimal rate. Therefore, while optimal translation of a yeast mRNA requires a leader length of at least some 30 nt, shorter leaders still allow considerable translation to take place.

Base Sequence↗

PubMed1999

Multiple 5'-untranslated exons in the nuclear respiratory factor 1 gene span 47 kb and contribute to transcript heterogeneity and translational efficiency.

Nuclear respiratory factor 1 (NRF-1) is a nuclear transcription factor that has been implicated in the nuclear control of respiratory chain expression in mammalian cells. Here, we demonstrate that a complex pattern of alternative splicing contributes to sequence heterogeneity within the human NRF-1 5'-untranslated region (UTR). At least six different 5'-UTR exons (UTRs 1-6) were detected in NRF-1 transcripts. These exons were mapped to human NRF-1 genomic clones and their sequences, including donor and acceptor splice junctions, determined. Two of the human UTR exons were derived from insertions of Alu-sq family members into the NRF-1 locus. The distance between the transcription initiation sites in UTR1 and the first protein coding exon is approx. 47kb, bringing the total length of the human NRF-1 gene to approx. 104kb. In contrast to human, only two UTR exons were found in mouse. The mouse UTR1 sequence obtained is identical to human UTR1, but mouse UTR2 bears no resemblance to any of the human exons. Mutations within human UTR1 modulate NRF-1 expression by interfering with mRNA translational efficiency in transfected cells and in an in vitro translation system. The effects of the mutations are proportional to their ability to disrupt predicted mRNA secondary structures within UTR1. Thus, the unusually high sequence conservation within UTR1 in part reflects selective constraints on translational expression.

5' Untranslated Regions↗

PubMed2002

Differential distribution and internal translation efficiency of hepatitis C virus quasispecies present in dendritic and liver cells.

Hepatitis C virus (HCV) is predominantly a hepatotropic virus. Nonetheless, there is mounting evidence that hematopoietic cells may support HCV replication. The HCV 5' untranslated region (5'UTR), responsible for initiation of viral translation, via an internal ribosome entry site (IRES), has been previously described to contain specific nucleotide substitutions when cultured in infected lymphoid cells. Our purpose was to establish whether the 5'UTR polymorphism of quasispecies from 3 cell compartments (liver, peripheral blood mononuclear cells [PBMG], and monocyte-derived dendritic cells [DCs]) of a patient chronically infected with HCV1b affects the corresponding translational efficiencies and thus the capacity for replication. The 5'UTR polymorphism was characterized by identification of changes at 3 crucial sites as compared with the reference nucleotide (nt) sequence: a G insertion between positions 19 and 20, a C>A substitution at position 204 and a G>A substitution at position 243. The quasispecies detected in DCs was unique and differed from those present in the liver, suggesting a particular tropism of HCV quasispecies for DCs. Moreover, its translational activity was significantly impaired when compared with those from liver and PBMCs in different cell lines. This impairment was thoroughly confirmed in primary cultures of both human hepatocytes and monocyte-derived DCs. Taken together, our data lend support both to a specific location and impaired replication of HCV quasispecies in DCs, which could be related to viral persistence and perturbation of DC function in chronically infected patients.

5' Untranslated Regions↗

PubMed2002

The 3' untranslated region of the new rat synaptic vesicle protein 2B mRNA transcript inhibits translational efficiency.

Post-transcriptional regulatory mechanisms have been shown to play a major role in gene expression in eukaryotic cells. Sequences within the 3'-untranslated region (3'UTR) of mRNAs have been described as being important for enhancing or inhibiting message translation. Using fluorescence microscopy, Western blotting and the reverse transcription-polymerase chain reaction (RT-PCR) method, we demonstrate that the 3'UTR of the new rat transcript of synaptic vesicle protein 2B (SV2Bb) mRNA is involved in post-transcriptional regulation of SV2Bb translation. When fused to a reporter gene, this 3'UTR markedly inhibited protein synthesis in transiently transfected cells and this decreased translational efficiency did not occur through changes in mRNA stability. In conclusion, our study gives new insights into unraveling the molecular mechanisms involved in the post-transcriptional regulation of the SV2B gene.

3' Untranslated Regions↗

PubMedDate not supplied

Codon usage and tRNA genes in eukaryotes: correlation of codon usage diversity with translation efficiency and with CG-dinucleotide usage as assessed by multivariate analysis.

The species-specific diversity of codon usage in five eukaryotes (Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and Homo sapiens) was investigated with principal component analysis. Optimal codons for translation were predicted on the basis of tRNA-gene copy numbers. Highly expressed genes, such as those encoding ribosomal proteins and histones in S. pombe, C. elegans, and D. melanogaster, have biased patterns of codon usage which have been observed in a wide range of unicellular organisms. In S. pombe and C. elegans, codons contributing positively to the principal component with the largest variance (Z1-parameter) corresponded to the optimal codons which were predicted on the basis of tRNA gene numbers. In D. melanogaster, this correlation was less evident, and the codons contributing positively to the Z1-parameter corresponded primarily to codons with a C or G in the codon third position. In X. laevis and H. sapiens, codon usage in the genes encoding ribosomal proteins and histones was not significantly biased, suggesting that the primary factor influencing codon-usage diversity in these species is not translation efficiency. Codon-usage diversity in these species is known to reflect primarily isochore structures. In the present study, the second additional factor was explained by the level of use of codons containing CG-dinucleotides, and this is discussed with respect to transcription regulation via methylation of CG-dinucleotides, which is observed in mammalian genomes.

Animals↗

PubMed2005

The influence of nucleotide sequences at and near ribosome-binding site on translational efficiency of the Bacillus subtilis rho gene.

The Bacillus subtilis rho gene encodes the transcription termination factor Rho that is produced at a low level in B. subtilis cells. No typical Shine-Dalgarno (SD) sequence lies at an appropriate distance from the translational start site of rho. However, the nucleotide sequence GTGGTG present upstream of the rho translational start site is highly conserved among rho genes of Bacilli. Base substitutions at the central GG or its downstream T abolished expression of rho-lacZ translational fusion, suggesting their importance in rho expression. Mutations at the relatively conserved sequence AAAG located further upstream of GTGGTG could also affect translational efficiency. Moreover, insertion of two or three nucleotides between these two conserved regions abrogated rho-lacZ expression, suggesting that the spacing is important. The possibility that the rho gene may contain a split SD sequence is discussed.

5' Untranslated Regions↗

PubMed1991

Translational efficiency and competitive ability of mRNAs with 5'-untranslated alpha beta-leader of potato virus X RNA.

The 5'-untranslated leader sequence of potato virus X (PVX) RNA (63 nucleotides apart from cap-structure) consists of two sub-sequences referred to as alpha-sequence (41 nucleotides with no G) and beta-sequence (42 nucleotides upstream from the first AUG). Computer-based folding predictions suggest that the 5'-proximal region of alpha beta-leader is unstructured. The second structural feature of alpha beta-leader is the presence of the sequences apparently complementary to the 3'-terminal region of 18S rRNA. The alpha beta-leader has been shown to strongly enhance the translation of the contiguous foreign gene (NPT1) transcripts in cell-free translation systems from rabbit reticulocytes (RRL), wheat germ (WG) and Krebs-2 ascite cell extract (KA). In competitive translation PVX, RNA strongly inhibited tobacco mosaic virus (TMV) RNA, in RRL and WG systems. No competition occurred between PVX and TMV RNAs in KA system. There was no correlation between the translational efficiency and competitive ability of PVX RNA in different cell-free translation systems. The competitive ability did not solely depend on the presence of alpha beta-leader in mRNA. We present evidence to suggest that alpha beta-leader together with about 150 bases of the coding sequence is responsible for the translation competitive ability of PVX RNA.

Base Sequence↗

PubMed1988

Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5' noncoding region.

Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction.

Animals↗

PubMed1995

Translational efficiency of gonadotropin-releasing hormone messenger ribonucleic acid is negatively regulated by phorbol ester in GT1-7 cells.

Our laboratory and others have reported that treatment of GT1-7 cells with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), inhibits transcription of the pro-GnRH gene and decreases messenger RNA (mRNA) levels. We were interested in whether translation of the existing GnRH mRNA decreases in parallel with these other indexes of biosynthesis after PMA treatment. GT1-7 cells were treated with PMA (100 nM) or vehicle for 0, 1, or 4 h. The cytosolic ribosome-associated RNA was isolated and layered on a continuous (10-40%) sucrose gradient, and fractions were analyzed for the distribution of ribosome-associated GnRH mRNA through the gradient by ribonuclease protection assay. The mRNA found in the lighter fractions is associated with fewer ribosomes per RNA, suggesting that these fractions are translated less efficiently, and RNA recovered from heavier fractions has a higher number of ribosomes per mRNA, representing mRNA that is more actively translated. We found that the distribution of the ribosome-associated GnRH mRNA was shifted into lighter fractions (i.e. fewer ribosomes per mRNA) after PMA treatment, indicating that a decrease in the translational efficiency of GnRH mRNA occurs after PMA treatment. Thus, PMA exerts inhibitory effects on translation of GnRH mRNA as well as on gene transcription, mRNA stability, and mRNA levels.

Animals↗

PubMed2001

The 3'-untranslated region of murine cyclooxygenase-2 contains multiple regulatory elements that alter message stability and translational efficiency.

Renal mesangial cells regulate their expression of the pro-inflammatory gene cyclooxygenase-2 (COX-2) through mechanisms involving gene transcription and post-transcriptional events. Post-transcriptional regulation of COX-2 is dependent, in part, on sequences within the 3'-untranslated region (3'-UTR) of the COX-2 mRNA. Insertion of the entire 3'-UTR of COX-2 into the 3'-UTR of luciferase resulted in a 70% decrease in luciferase enzymatic activity. Measurement of steady-state reporter gene mRNA levels suggested that the loss of activity was due to decreased translational efficiency. Deletion analysis identified the first 60 nucleotides of the 3'-UTR of COX-2 as a major translational control element. This region of the 3'-UTR of COX-2 is highly conserved across species; is AU-rich; and contains multiple repeats of the regulatory sequence AUUUA, reported to confer post-transcriptional control. In addition, we identified regions of the 3'-UTR of COX-2 outside of the first 60 nucleotides that altered message stability. Some of these regions contained AUUUA consensus sequences, whereas others did not, and represent novel control elements. These results suggest that expression of COX-2 in mesangial cells depends on the complex integration of multiple signals derived from the 3'-UTR of the message.

3' Untranslated Regions↗

PubMed1997

In vivo translational efficiency of different hepatitis C virus 5'-UTRs.

Initiation of translation in hepatitis C virus (HCV) is dependent on the presence of an internal ribosome entry site (IRES) contained in its 341-nt-long 5'-untranslated region (UTR). This region is very conserved among different isolates and has been used to classify HCV isolates in six different genotypes. These genotypes differ in nucleotide sequence that generally preserve the IRES structure. However, the small differences seen may be biologically and clinically significant as the HCV strains seem to differ from each other in several important ways, such as the behaviour of the viral infection and the response to interferon therapy. Therefore, differences in translational initiation efficiency amongst the various genotypes could provide an explanation for these phenomena. Using a bicistronic expression system we have compared the in vivo translational ability of the three most common European genotypes of HCV (1, 2, and 3). The results show that genotype 3 is less able than 1 and 2 to promote translation initiation. In addition, by site-directed mutagenesis of the sequence of the IRES domain III apical stem loop structure, we have shown that the conservation of the primary nucleotide sequence and not only the structure, is important for the conservation of IRES function.

Animals↗

PubMed2005

The translational efficiencies of the two Leishmania infantum HSP70 mRNAs, differing in their 3'-untranslated regions, are affected by shifts in the temperature of growth through different mechanisms.

Exposure of Leishmania promastigotes to the temperature of their mammalian hosts induces a typical heat-shock response. In Leishmania infantum, HSP70 is encoded by two types of genes that differ in their 3'-untranslated regions (3'-UTRs). Previously, we have shown that specific transcripts for each gene are present in promastigotes growing at normal temperature (26 degrees C), but only transcripts with 3'-UTR-type I (3'-UTRI) accumulate in a temperature-dependent manner. Here, we have investigated the translational efficiencies of both types of HSP70 transcripts at the different temperatures that the parasite encounters in the insect (26 degrees C, normal temperature) or in the mammalian host (heat-shock temperatures). Interestingly, 3'-UTRI-bearing transcripts (HSP70-I) were found associated with ribosomes in promastigotes at normal and heat-shock temperatures, whereas the HSP70-II transcripts appear to be preferentially translated at heat-shock temperatures but not at 26 degrees C. We have analyzed the function of these UTRs in the translational control by use of plasmid constructs in which the CAT reporter gene was flanked by UTRs of the HSP70 genes. Unexpectedly, it was found that CAT transcripts with 3'-UTRII bind to ribosomes at 26 degrees C, and, indeed, the CAT protein is synthesized. A valid conclusion of these experiments was that both types of 3'-UTRs are essential for translation of HSP70 mRNAs at heat shock temperatures, although the 3'-UTRII is more efficient during severe heat shock (39 degrees C). In addition, these results suggest that sequence region other than the 3'-UTR of HSP70-II gene is involved in the translational silent state of HSP70-II transcripts at 26 degrees C. Finally, a null mutant has been created by targeted disruption of both HSP70-II alleles. Remarkably, the deltaHSP70 mutant synthesizes HSP70 at a lower rate than the wild-type parasites. Overall, our data suggest that the biological function of the HSP70-II gene is to top up HSP70 levels under conditions of stress.

3' Untranslated Regions↗