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PubMed1998

Translating health status questionnaires and evaluating their quality: the IQOLA Project approach. International Quality of Life Assessment.

This article describes the methods adopted by the International Quality of Life Assessment (IQOLA) project to translate the SF-36 Health Survey. Translation methods included the production of forward and backward translations, use of difficulty and quality ratings, pilot testing, and cross-cultural comparison of the translation work. Experience to date suggests that the SF-36 can be adapted for use in other countries with relatively minor changes to the content of the form, providing support for the use of these translations in multinational clinical trials and other studies. The most difficult items to translate were physical functioning items, which used examples of activities and distances that are not common outside of the United States; items that used colloquial expressions such as pep or blue; and the social functioning items. Quality ratings were uniformly high across countries. While the IQOLA approach to translation and validation was developed for use with the SF-36, it is applicable to other translation efforts.

Cross-Cultural Comparison↗

PubMed2004

The MeSH translation maintenance system: structure, interface design, and implementation.

The National Library of Medicine (NLM) produces annual editions of the Medical Subject Headings (MeSH). Translations of MeSH are often done to make the vocabulary useful for non-English users. However, MeSH translators have encountered difficulties with entry vocabulary as they maintain and update their translation. Tracking MeSH changes and updating their translations in a reasonable time frame is cumbersome. NLM has developed and implemented a concept-centered vocabulary maintenance system for MeSH. This system has been extended to create an interlingual database of translations, the MeSH Translation Maintenance System (MTMS). This database allows continual updating of the translations, as well as facilitating tracking of the changes within MeSH from one year to another. The MTMS interface uses a Web-based design with multiple colors and fonts to indicate concepts needing translation or review. Concepts for which there is no exact English equivalent can be added. The system software encourages compliance with the Unicode standard in order to ensure that character sets with native alphabets and full orthography are used consistently.

Databases as Topic↗

PubMed2003

Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.

Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells.

Animals↗

PubMed2025

Translation of the downstream ORF from bicistronic mRNAs by human cells: Impact of codon usage and splicing in the upstream ORF.

Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the "main" ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery. Here, we introduce an experimental model to study gene expression regulation from virus-like bicistronic mRNAs in human cells. The model consists of a short upstream ORF and a reporter downstream ORF encoding a fluorescent protein. We have engineered synonymous variants of the upstream ORF to explore large parameter space, including codon usage preferences, mRNA folding features, and splicing propensity. We show that human translation machinery can translate the downstream ORF from bicistronic mRNAs, albeit reporter protein levels are thousand times lower than those from the upstream ORF. Furthermore, synonymous recoding of the upstream ORF exclusively during elongation significantly influences its own translation efficiency, reveals cryptic splice signals, and modulates the probability of downstream ORF translation. Our results are consistent with a leaky scanning mechanism facilitating downstream ORF translation from bicistronic mRNAs in human cells, offering new insights into the role of upstream ORFs in translation regulation.

Humans↗

PubMed1997

In vitro mutational and inhibitory analysis of the cis-acting translational elements within the 5' untranslated region of coxsackievirus B3: potential targets for antiviral action of antisense oligomers.

The 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) RNA forms a highly ordered secondary structure that has been implicated in controlling initiation of viral translation by internal ribosomal entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the 5'UTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron, the chloramphenicol acetyltransferase gene, designed to block ribosomal scanning, the CVB3 5'UTR was capable of directing the internal initiation of translation of the downstream reporter gene (P1), confirming the presence of an internal ribosomal entry site (IRES). This finding was further supported by the data on predicted secondary structures within the 5'UTR. Of special note, analysis of various deletion mutants demonstrated that the IRES of CVB3 is located roughly at stem-loops G, H, and I spanning nucleotides (nt) 529 and 630. The region from nt 1 to 63 (stem-loop A) also appears important, and it may be an essential binding site for translation initiation factors. Based on these findings, in vitro translation inhibition assays using RNA fragments of the 5'UTR as inhibitor were performed. Both antisense and sense RNA segments transcribed from these two cis-acting regions and the surrounding sequence of the initiation codon AUG showed strong inhibition of viral protein synthesis. Antisense molecules may inhibit translation by blocking ribosome and initiation factor binding within the 5'UTR via specific hybridization to their viral RNA target sequences, while sense sequences may function by competing with viral RNA for ribosomes and/or translation initiation factors. These cis-acting translational elements may serve as potential targets for the antiviral action of oligomers.

Animals↗

PubMed2001

Construction of regulatable picornavirus IRESes as a test of current models of the mechanism of internal translation initiation.

Picornavirus internal ribosome entry sites (IRESs) are approximately 450 nt. RNA elements that direct internal initiation of translation, such that when placed between the two cistrons of a dicistronic construct, they drive independent translation of the downstream cistron. Consequently they have been widely used for coordinated expression of two or more proteins. All picornavirus IRESs have an AUG triplet at the very 3' end, which is thought to be the actual site of internal ribosome entry. However with some IRESs, such as foot-and-mouth disease virus, and especially poliovirus, the majority of ribosomes do not initiate translation at this putative entry site AUG, but at the next AUG further downstream, which is thought to be accessed by a process of linear ribosome scanning from the entry site. If this is so, then it should be possible to regulate IRES-dependent translation by inserting an iron responsive element (IRE) between the putative entry site AUG and the main functional initiation site. This should make IRES-dependent translation sensitive to the concentration of iron regulatory protein (IRP), the protein that specifically binds to the IRE. This has been attempted with both the foot-and-mouth disease virus and poliovirus IRESs, and was successful in so far as an inhibition specifically of IRES-dependent translation was observed that was strictly dependent on both the presence of IRP and of a functional IRE motif inserted in the sense orientation. However, the range over which expression could be varied was rather limited (three- to fourfold maximum), because some IRES-dependent translation remained completely refractory to inhibition by even very high IRP concentrations. In contrast, with a cap-proximal IRE in the 5' untranslated region of an mRNA translated by the scanning mechanism, addition of sufficient IRP results in complete inhibition. These results support the model of IRES-promoted ribosome entry at an upstream site followed by strictly linear scanning to the main functional initiation site for the majority of internal initiation events, but imply that some ribosomes must access the functional initiation site by another route, possibly a nonlinear shunting-like mechanism.

Aphthovirus↗

PubMed2003

Use of differential item functioning analysis to assess the equivalence of translations of a questionnaire.

In cross-national comparisons based on questionnaires, accurate translations are necessary to obtain valid results. Differential item functioning (DIF) analysis can be used to test whether translations of items in multi-item scales are equivalent to the original. In data from 10,815 respondents representing 10 European languages we tested for DIF in the nine translations of the EORTC QLQ-C30 emotional function scale when compared to the original English version. We tested for DIF using two different methods in parallel, a contingency table method and logistic regression. The DIF results obtained with the two methods were similar. We found indications of DIF in seven of the nine translations. At least two of the DIF findings seem to reflect linguistic problems in the translation. 'Imperfect' translations can affect conclusions drawn from cross-national comparisons. Given that translations can never be identical to the original we discuss how findings of DIF can be interpreted and discuss the difference between linguistic DIF and DIF caused by confounding, cross-cultural differences, or DIF in other items in the scale. We conclude that testing for DIF is a useful way to validate questionnaire translations.

Adult↗

PubMed2026

Unbiased screen of human transcriptome reveals an unexpected role of 3'UTRs in translation initiation.

Although most eukaryotic mRNAs require a 5'-cap for translation initiation, some can also be translated through a poorly studied cap-independent pathway. Here we develop a circRNA-based system and unbiasedly identify more than 10,000 sequences in the human transcriptome that contain Cap-independent Translation Initiators (CiTIs). Surprisingly, most of the identified CiTIs are located in 3'UTRs, which mainly promote translation initiation in mRNAs bearing highly structured 5'UTR. Mechanistically, CiTI recruits several translation initiation factors including eIF3 and DHX29, which in turn unwind 5'UTR structures and facilitate ribosome scanning. Functionally, we show that the translation of HIF1A mRNA, an endogenous DHX29 target, is antagonistically regulated by its 5'UTR structure and a new 3'-CiTI in response to hypoxia. Consistently, deletion of 3'-CiTI suppresses cell growth in hypoxia and tumor progression in vivo. Collectively, our study uncovers a new regulatory mode for translation where the 3'UTR actively participate in the translation initiation.

Humans↗

PubMed2006

Different modes of translation for hid, grim and sickle mRNAs in Drosophila.

Protein synthesis is inhibited during apoptosis. However, the translation of many mRNAs still proceeds driven by internal ribosome entry sites (IRESs). Here we show that the 5'UTR of hid and grim mRNAs promote translation of uncapped-mRNA reporters in cell-free embryonic extracts and that hid and grim mRNA 5'UTRs drive IRES-mediated translation. The translation of capped-reporters proceeds in the presence of cap competitor and in extracts where cap-dependent translation is impaired. We show that the endogenous hid and grim mRNAs are present in polysomes of heat-shocked embryos, indicating that cap recognition is not required for translation. In contrast, sickle mRNA is translated in a cap-dependent manner in all these assays. Our results show that IRES-dependent initiation may play a role in the translation of Drosophila proapoptotic genes and suggest a variety of regulatory pathways.

5' Untranslated Regions↗

PubMed2003

Translating and validating the Finnish version of the Manchester Clinical Supervision Scale.

Evaluation research provides new perspectives for clinical supervision (CS), and international collaboration offers advantages to develop valid instruments for this purpose. Besides translation, an instrument developed and tested in another culture requires systematic validation. The study focuses on the translation process of the Manchester Clinical Supervision Scale for testing in Finland carried out collaboratively between the Universities of Tampere and Manchester. The instrument is a 45-item questionnaire with a Likert-type (1-5) scale comprising seven sub-scales: trust and rapport, supervisor advice and support, improved care and skills, importance and value of CS, finding time, personal issues and reflection and total score. At first, a licensed translator translated the instrument into Finnish. A native British language teacher at the University language centre performed the blind back-translation into English. The translations were compared by both collaborative parties and by three experienced Finnish supervisors. A pilot sample (n = 182) was collected to test the translated instrument. In this sample Cronbach's alpha value for the total score was 0.9227 and in the sub-scales 0.6393-0.8838. The mean values in the sub-scales were 14.2-29.3, SDs 3.02-3.88 and modes 14.0-30.0. The British test sample had almost similar values. Translating an instrument into another language not only requires expertise in language, but also in practice. The cultural validation is the most important phase in the process that can be accomplished with pilot testing and statistical methods. However, further expert evaluation is required for the validity of the instrument.

Clinical Competence↗

PubMed2002

An intrinsic control element for translational initiation in class 1 integrons.

Integrons are genetic elements able to capture anti-biotic resistance and other genes and to promote their transcription. Here, we have investigated integron-dependent translation of an aminoglycoside 6'-N-acetyltransferase gene (aac(6')-Ib7) inserted at the attI1 site. N-terminal sequencing revealed that translation of this gene was initiated at a GTG codon, which is not part of a plausible translation initiation region (TIR). A short open reading frame (called ORF-11) overlapping the attI1 site was probed by site-directed mutagenesis for its contribution to aac(6')-Ib7 translation. When ORF-11 and its TIR were deleted en bloc, translational efficiency dropped by over 80%, as determined with an acetyltransferase- luciferase fusion product. Invalidation of the ATG start codon of ORF-11 or its putative Shine-Dalgarno sequence resulted in a decrease of over 60%, whereas the decrease was much less pronounced when the amino acid sequence of the putative ORF-11-encoded peptide was altered or when the distance between ORF-11 and aac(6')-Ib7 was doubled. This demonstrates that aac(6')-Ib7 translation is dependent upon the translation of ORF-11, but almost certainly not upon the corresponding peptide. These results lead us to conclude that an intrinsic short ORF present in the 5'-conserved segment of many class 1 integrons may substantially enhance expression at the translational level of captured TIR-deficient anti-biotic resistance genes.

Amino Acid Sequence↗

PubMed2003

Evidence for the translation initiation of leaderless mRNAs by the intact 70 S ribosome without its dissociation into subunits in eubacteria.

In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5'-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA.70 S.fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process.

Escherichia coli↗

PubMed1997

Cap-independent leaky scanning as the mechanism of translation initiation of a plant viral genomic RNA.

The genome of plum pox virus contains a single open reading frame that is translated into a large polyprotein. Although the open reading frame starts at nucleotide 36 (36AUG), it is translated from the second, 147AUG, which is in a more favourable context for translation initiation. We have carried out in vitro translation and transient expression analysis in protoplasts of a nested set of substitution and deletion mutants, and the results show that no internal structure in the 5' noncoding region of plum pox virus is necessary for efficient translation initiation. On the other hand, when the cryptic 36AUG was placed in a favourable context, it turned into an efficient initiation codon in vitro. Furthermore, AUGs that were placed in a favourable context, initiating short intraleader open reading frames, repressed translation initiation from the 147AUG in vitro and in vivo. These results point to leaky scanning as the mechanism of translation initiation of plum pox virus RNA. Nevertheless, it is a peculiar leaky scanning where the initiation of translation does not require a cap structure at the 5' end. This fact is congruent with the experimentally predicted absence of a stable secondary structure at the 5' noncoding region.

Animals↗

PubMed2001

Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'.

The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A 'primary' intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational 'cleavage' products quantified. The processing products from our artificial polyprotein systems showed a molar excess of 'cleavage' product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and beta-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNA(Gly) ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal 'skip' from one codon to the next without the formation of a peptide bond.

Animals↗

PubMed2000

Biogenesis of the chloroplast-encoded D1 protein: regulation of translation elongation, insertion, and assembly into photosystem II.

Regulation of translation elongation, membrane insertion, and assembly of the chloroplast-encoded D1 protein of photosystem II (PSII) was studied using a chloroplast translation system in organello. Translation elongation of D1 protein was found to be regulated by (1) a redox component that can be activated not only by photosynthetic electron transfer but also by reduction with DTT; (2) the trans-thylakoid proton gradient, which is absolutely required for elongation of D1 nascent chains on the thylakoid membrane; and (3) the thiol reactants N-ethylmaleimide (NEM) and iodosobenzoic acid (IBZ), which inhibit translation elongation with concomitant accumulation of distinct D1 pausing intermediates. These results demonstrate that D1 translation elongation and membrane insertion are tightly coupled and highly regulated processes in that proper insertion is a prerequisite for translation elongation of D1. Cotranslational and post-translational assembly steps of D1 into PSII reaction center and core complexes occurred independently of photosynthetic electron transfer or trans-thylakoid proton gradient but were strongly affected by the thiol reactants DTT, NEM, and IBZ. These compounds reduced the stability of the early PSII assembly intermediates, hampered the C-terminal processing of the precursor of D1, and prevented the post-translational reassociation of CP43, indicating a strong dependence of the D1 assembly steps on proper redox conditions and the formation of disulfide bonds.

Carotenoids↗

PubMed2006

Translation of the short version of the Perinatal Grief Scale into Swedish.

INTRODUCTION: Women's emotions and grief after miscarriage are influenced not only by the context in which the miscarriage occurred but also by their past experience, the circumstances around the miscarriage and their future prospects. Their emotions therefore express a specific form of grief. Normally the time needed to work through the loss varies. A number of different scales, measuring women's emotions and grief after miscarriage have been published. One instrument that measures the specific grief, such as the grief after miscarriage is the Perinatal Grief Scale (PGS) that was designed to measure grief after perinatal loss and has good reliability and validity. AIMS: The purpose of this study was to translate the PGS into Swedish and to use the translation in a small pilot study. MATERIAL AND METHOD: The original short version of the PGS was first translated from English into Swedish and then back-translated into English, using different translators. During translation and back-translation, not only the linguistic and grammatical aspects were considered but also cultural differences. The Likert 5-point and a 10-point scale were tested in a pilot study where 12 volunteers anonymously answered the PGS twice. The intra-personal correlations were compared and analysed with weighted kappa-coefficient. FINDINGS: In all, five different versions were tested before the final Swedish version was established. The weighted kappa-coefficient for the volunteers was 0.58, which is regarded as representing good reproducibility. CONCLUSION: The PGS was translated successfully into Swedish and could be used in a Swedish population. As this work is rather time-consuming we therefore wish to publish the Swedish version so that it may be used by other researchers.

Abortion, Spontaneous↗

PubMed1998

Coding sequences enhance internal initiation of translation by hepatitis A virus RNA in vitro.

Hepatitis A virus (HAV), unlike other picornaviruses, has a slow-growth phenotype in permissive cell lines and in general does not induce host cell cytopathology. Although there are no published reports of productive infection of HeLa cells by HAV, HAV RNA appears to be readily translated in HeLa cells when transcribed by T7 RNA polymerase provided by a recombinant vaccinia virus. The 5' noncoding region of HAV was fused to poliovirus (PV) coding sequences to determine the effect on translation efficiency in HeLa cell extracts in vitro. Conditions were optimized for utilization of the HAV internal ribosome entry segment (IRES). Transcripts from chimeric constructs fused precisely at the initiation codon were translated very poorly. However, chimeric RNAs which included 114 or more nucleotides from the HAV capsid coding sequences downstream of the initiation codon were translated much more efficiently than those lacking these sequences, making HAV-directed translation efficiency similar to that directed by the PV IRES. Sixty-six nucleotides were insufficient to confer increased translation efficiency. The most 5'-terminal HAV 138 nucleotides, previously determined to be upstream of the IRES, had an inhibitory effect on translation efficiency. Constructs lacking these terminal sequences, or those in which the PV 5'-terminal sequences replaced those from HAV, translated three- to fourfold better than those with the intact HAV 5'-terminal end.

Cell Extracts↗

PubMed2002

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1.

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.

Adaptor Proteins, Signal Transducing↗