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PubMed2000

Translation initiation of the insulin-like growth factor I receptor mRNA is mediated by an internal ribosome entry site.

The insulin-like growth factor I receptor (IGF-IR) is a heterotetrameric receptor mediating the effects of insulin-like growth I and other growth factors. This receptor is encoded by an mRNA containing an unusually long, G-C-rich, and highly structured 5' untranslated region. Using bicistronic constructs, we demonstrated here that the 5' untranslated region of the IGF-IR allows translation initiation by internal ribosome entry and therefore constitutes an internal ribosome entry site. In vitro cross-linking revealed that this internal ribosome entry site binds a protein of 57 kDa. Immunoprecipitation of UV cross-linked proteins proved that this protein was the polypyrimidine tract-binding protein, a well known regulator of picornavirus mRNA translation. The efficiency of translation of the endogenous IGF-IR mRNA is not affected by rapamycin, which is a potent inhibitor of cap-dependent translation. This result provides evidence that the endogenous IGF-IR mRNA is translated, at least in part, through a cap-independent mechanism. This is the first report of a growth factor receptor containing sequence elements that allow translation initiation to occur by internal initiation. Because the IGF-IR has a pivotal function in the cell cycle, this mechanism of translation regulation could play a crucial role in the control of cell proliferation and differentiation.

5' Untranslated Regions↗

PubMed2004

Translation initiation with 70S ribosomes: an alternative pathway for leaderless mRNAs.

It is generally accepted that translation in bacteria is initiated by 30S ribosomal subunits. In contrast, several lines of rather indirect in vitro evidence suggest that 70S monosomes are capable of initiating translation of leaderless mRNAs, starting with the A of the initiation codon. In this study, we demonstrate the proficiency of dedicated 70S ribosomes in in vitro translation of leaderless mRNAs. In support, we show that a natural leaderless mRNA can be translated with crosslinked 70S wild-type ribosomes. Moreover, we report that leaderless mRNA translation continues under conditions where the prevalence of 70S ribosomes is created in vivo, and where translation of bulk mRNA ceases. These studies provide in vivo as well as direct in vitro evidence for a 70S initiation pathway of a naturally occurring leaderless mRNA, and are discussed in light of their significance for bacterial growth under adverse conditions and their evolutionary implications for translation.

5' Untranslated Regions↗

PubMed2005

UNR translation can be driven by an IRES element that is negatively regulated by polypyrimidine tract binding protein.

Upstream of N-ras (Unr) has been described as an internal initiation trans-acting factor (ITAF) in the cap-independent translation of some particular viral and cellular mRNAs. Two factors led us to hypothesize that the UNR 5'-untranslated region (5'-UTR) may contain an internal ribosome entry site (IRES). The first was the requirement for persisting Unr expression under conditions that correlate with cap-independent translation. The other was the observation that the primary UNR transcript contains a 447 nt long 5'-UTR including two upstream AUGs that may restrict translation initiation via cap-dependent ribosome scanning. Here we report that the UNR 5'-UTR allows IRES-dependent translation, as revealed by a dicistronic reporter assay. Various controls ruled out the contribution of leaky scanning, cryptic promoter sequences or RNA processing events to the ability of the UNR 5'-UTR to mediate internal initiation of translation. Ultraviolet cross-linking analysis and RNA affinity chromatography revealed the binding of polypyrimidine tract binding protein (PTB) to the UNR IRES, requiring a pyrimidine-rich region (nucleotides 335-355). Whereas overexpression of PTB in several cell lines inhibited UNR IRES activity and UNR protein expression, depletion of endogenous PTB using RNAi increased UNR IRES activity. Moreover, a mutant version of the UNR IRES lacking the PTB binding site was more efficient at directing IRES-mediated translation. In conclusion, our results demonstrate that translation of the ITAF Unr can itself be regulated by an IRES that is downregulated by PTB.

5' Untranslated Regions↗

PubMed2005

Functional analysis of factors involved in trans-translation.

trans-translation is a rescue system for stalled ribosomes to terminate the translation for recycling of ribosome and lead the incomplete protein on the stalled ribosome to degradation. In trans-translation, several proteins and RNAs are involved. tmRNA and SmpB are only factors known to be essential for trans-translation. EF-Tu and EF-G functions in translation and also are seemed to function in trans-translation. Ribosomal protein S1 is known to bind tmRNA. In this study, we cloned the gene of tmRNA from Thermus thermophilus and analyzed the functions of factors involved in trans-translation in vitro.

Base Sequence↗

PubMed2026

Involvement of tRNA thiolation in uORF-mediated translational regulation during Xylogenesis in Arabidopsis thaliana.

Post-transcriptional modification of tRNAs is an important mechanism for regulating translation efficiency and cellular homeostasis, yet its contribution to upstream open reading frame (uORF)-mediated translational control remains largely unexplored. In this study, we investigated the role of tRNA thiolation in thermospermine-dependent regulation of xylem development in Arabidopsis thaliana. Using a suppressor screen of the thermospermine-deficient mutant acaulis5 (acl5), which exhibits dwarfism and excessive xylem differentiation, we identified suppressor-of-acl502 (sac502) as a recessive loss-of-function allele of CTU2, a gene encoding a key enzyme in the biosynthesis of the wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine. Mutations in other components of the same modification pathway, including ROL5 and TRM9, similarly suppressed the acl5 phenotype. Translational analyses using 5' leader-GUS reporter constructs revealed that the ctu2 mutation did not enhance translation of the mRNA containing a thermospermine-responsive uORF of SAC51, but instead significantly reduced translation of that of SACL3, a member of the SAC51 family, and that of LONESOME HIGHWAY (LHW), which contains another conserved uORF in the 5' leader region. Polysome profiling further demonstrated decreased association of SACL3 and LHW mRNAs with actively translating ribosomes in ctu2. Genetic interaction analyses supported the conclusion that the suppression of excessive xylem formation in acl5 by ctu2 is attributable to reduced LHW activity. In addition, ctu2 mutants displayed increased sensitivity to exogenous thermospermine, resembling the response of lhw mutants. Together, our results reveal that tRNA thiolation contributes to uORF-mediated translational regulation of key developmental regulators and identify tRNA modification as an important regulatory layer controlling vascular development.

Arabidopsis↗

PubMed2002

Translating instruments into other languages: development and testing processes.

Cross-cultural influences affect perceptions and health practices, which are 2 areas of nursing concern. Culturally sensitive assessment instruments are needed, but many challenges exist in obtaining valid and reliable measurement. Translating questionnaires for cross-cultural research is fraught with methodological pitfalls related to colloquial phrases, jargon, idiomatic expressions, word clarity, and word meanings. It cannot be assumed that a particular concept has the same relevance across cultures. Simply translating an English version word-for-word into another language is not adequate to account for linguistic and cultural differences. Ideally, the perspectives of people from the culture about the concept of interest should be studied first, but often a practical alternative is to find and translate a tool developed in another culture. The purpose of this article is to describe important considerations in conducting translation for equivalence, types of equivalence, and strategies to translate instruments that promote equivalence and how to test the translated version for equivalence. These concepts and strategies are illustrated by describing the translation process of Hilton's Uncertainty Stress Scale into French and the use and testing of the French version with a French Canadian sample in Skrutkowski's study of perceived uncertainty in adult survivors of cancer.

Adult↗

PubMed1995

Developing a translation of the McGill pain questionnaire for cross-cultural comparison: an example from Norway.

The ability to measure pain across diverse cultures is important for understanding the universal aspects of pain and expediting nursing intervention. The McGill Pain Questionnaire (MPQ) is the most valid and reliable single multidimensional pain instrument available for measuring pain. Although it has been translated in several languages, most efforts, including two Norwegian translations, have resulted in a variety of new versions, all lacking sufficient faithfulness to the original MPQ to allow qualitative or quantitative cross-cultural comparisons. There is a need for direct translations that maintain the original denotation, connotation and numerical value of the MPQ and thus provide a base for future cross-cultural studies of pain. This paper reports on the development and initial testing of a direct translation of the McGill Pain Questionnaire into Norwegian (NMPQ). The translation process involved three phases: translation, back-translation and consensual. In order to evaluate its performance and validity, the NMPQ was administered to a group of adult surgical patients at two different points in time during the postoperative period. The NMPQ was examined for its feasibility, its sensitivity in detecting decreases in intensity of pain postoperatively and for construct validity. A visual analogue scale was used to check for converging validity, and Spielberger's state anxiety scale was used to assess discriminate validity. The initial testing of the NMPQ with adult surgical patients suggests that the NMPQ is culturally acceptable, relevant, sensitive to fluctuations in pain and numerically consistent with the original MPQ. The moderate levels of validity attend lend considerable assurance to the instruments readiness for use in cross-cultural studies of pain.

Adult↗

PubMed2005

Obesity: translation and testing of a survey instrument for use in Taiwan.

AIM AND OBJECTIVES: The purpose of this paper is to describe a cross-cultural translation and validation methodology recommended by Vallerand. The translation technique has seven steps and ensures the instrument provides data that are valid and reliable. BACKGROUND: Translation of data collection instruments is a challenge increasingly being faced by overseas students studying in the West as they choose to research contextual issues of relevance to their home culture. METHODS: Six of Vallerand's seven steps are used and modified as the methodical translation of a survey from English to Mandarin. RESULTS: The results revealed there was no significant difference between the original English questionnaire and Mandarin questionnaire. The validation study indicated good internal consistency. The construct validity of the Mandarin version was determined except one item which failed the test. The validation demonstrates only minor effects of language translation. CONCLUSION: The Mandarin version thus is a valid tool for investigating from the perspective of school nurses, their role and potential contribution to health promotion in relation to the prevention of obesity among school children in Taiwan. RELEVANCE TO CLINICAL PRACTICE: The results of the study will enable school nurses and their managers to reflect critically on their contribution to the prevention of obesity among Taiwanese school children. The translation technique has enabled the researcher to minimize errors that may have occurred in translation. It adds to the quality of the instrument and offers opportunities in the future to undertake cross-cultural comparison in an issue of great concern globally.

Attitude of Health Personnel↗

PubMed1988

Determinants of translational initiation efficiency in the atp operon of Escherichia coli.

Transcription and translation of the atp genes encoding the subunits b, delta, alpha, gamma and epsilon of the Escherichia coli H+-ATPase were studied. The nature and quantities of the respective transcripts initiated from different promoters were compared with overall expression rates thus yielding accurate information about relative translational efficiency and its coupling to mRNA levels. Part of the highly efficient subunit c gene translational initiation region (TIR) was used as a tool in manipulating the TIRs of the other genes. Rate control of atp cistron translation occurs at the initiation level and is determined locally by each gene's TIR. In this way, individual subunit synthesis rates are set to match the requirements for H+-ATPase assembly. There is no (or very restricted) translational coupling between the cistrons. Translational initiation rates of the normally weakly expressed atp genes could be increased by up to a factor of 27 by manipulating the sequences upstream of the start codons, despite biased codon usages. In the presence of an improved upstream sequence, the N-terminal sequence of the subunit gamma gene exerted a limiting effect. This could be relieved by altering the sequence of the first seven codons. The levels of subunit gamma mRNA were more sensitive to changes in translational efficiency than the concentrations of the other atp mRNAs. The relationships between initiation efficiency and primary and secondary structure in the natural and manipulated atp TIRs are discussed in detail.

Base Sequence↗

PubMed1989

Independent and coupled translational initiation of atp genes in Escherichia coli: experiments using chromosomal and plasmid-borne lacZ fusions.

The translational initiation rates directed by the translational initiation regions (TIRs) of the atpB, atpH, atpA and atpG genes of Escherichia coli were investigated using lacZ fusions present on plasmids as well as integrated into the chromosome. This was the first investigation of the translational efficiency of the atpB gene, whose unfused product (subunit a) can be toxic to the cell. The specific mRNA levels, rates of in vivo protein synthesis and beta-galactosidase activities encoded by the atp::lacZ fusions were compared in order to obtain valid estimates of relative translation rates. The results indicate that in the E. coli atp operon, translation directed by the atpB, atpH and atpG TIRs is less efficient than that directed by the atpA TIR, and are thus consistent with earlier measurements of direct atp gene expression. Initiation is, however, to differing extents, controlled by coupling to the translation of upstream neighbours. There is particularly tight coupling between atpH and atpA. Increasing the distance between these two genes whilst maintaining the original atpA TIR structure decreased the degree of coupling. The influence of manipulations of the atpG TIR structure upon translational efficiency was quantitatively more pronounced when the atpG fusions were present as a single copy per chromosome. This is likely to be related to the mRNA binding characteristics of 30S ribosomal subunits and/or to the influence of other (trans-acting) factors. The control of independent and coupled initiation at the atp TIRs is discussed in relation to mRNA structure and possible cis and trans regulatory phenomena.

Bacteriophage lambda↗

PubMed1994

Vitamin B12 repression of the cob operon in Salmonella typhimurium: translational control of the cbiA gene.

Expression of the cob operon is repressed by B12 via a post-transcriptional control mechanism which requires sequence elements within the leader region of the mRNA and the first gene of the operon, the cbiA gene. Here we show that B12 repression of cbiA gene expression occurs at the level of translation initiation through sequestration of the ribosomal binding site (rbs) in an RNA hairpin. Analysis of mutations that destabilize or restabilize the secondary structure demonstrates that folding of the hairpin is essential for repression. The existence of the hairpin was confirmed by a secondary structure analysis of RNA from the wild type and three mutants. Deletions that remove the upstream part of the leader confer a drastic reduction in translation efficiency. This low-level translation is caused by the hairpin, as indicated by the finding that suppressor mutations that destabilize the hairpin restore efficient translation. Thus, the native upstream RNA functions as a translation enhancer and acts to relieve the hairpin's inhibitory effect on translation initiation. The inhibitory effect of the hairpin was confirmed by a ribosomal toeprinting analysis. We propose that the translational control of the cbiA gene mediates repression of the entire cob operon.

Base Sequence↗

PubMed2004

Translation repression by an RNA polymerase elongation complex.

Bacteriophage lambda N and bacterial Nus proteins together with a unique site NUT in the leader of the early viral N gene transcript bind RNA polymerase (RNAP) and form a highly processive antitermination complex; N bound at NUT also represses N translation. In this study, we investigate whether N and NUT cause N translation repression as part of the antitermination complex by testing conditions that inhibit the formation of the N-modified transcription complex for their effect on N-mediated translation repression. We show that nus and nut mutations that in combination destabilize multiple interactions in the antitermination complex prevent N-mediated translation repression. Likewise, transcription of the nut-N region by T7 RNAP, which does not lead to the assembly of an effective antitermination complex when N is supplied, eliminates translation repression. We also demonstrate that a unique mutant beta subunit of RNAP reduces N-mediated translation repression, and that overexpression of transcription factor NusA suppresses this defect. We conclude that the N-modified RNAP transcription complex is necessary to repress N translation.

Base Sequence↗

PubMed2006

Resistance of mRNA translation to acute endoplasmic reticulum stress-inducing agents in herpes simplex virus type 1-infected cells requires multiple virus-encoded functions.

Via careful control of multiple kinases that inactivate the critical translation initiation factor eIF2 by phosphorylation of its alpha subunit, the cellular translation machinery can rapidly respond to a spectrum of environmental stresses, including viral infection. Indeed, virus replication produces a battery of stresses, such as endoplasmic reticulum (ER) stress resulting from misfolded proteins accumulating within the lumen of this organelle, which could potentially result in eIF2alpha phosphorylation and inhibit translation. While cellular translation is exquisitely sensitive to ER stress-inducing agents, protein synthesis in herpes simplex virus type 1 (HSV-1)-infected cells is notably resistant. Sustained translation in HSV-1-infected cells exposed to acute ER stress does not involve the interferon-induced, double-stranded RNA-responsive eIF2alpha kinase PKR, and it does not require either the PKR inhibitor encoded by the Us11 gene or the eIF2alpha phosphatase component specified by the gamma(1)34.5 gene, the two viral functions known to regulate eIF2alpha phosphorylation. In addition, although ER stress potently induced the GADD34 cellular eIF2alpha phosphatase subunit in uninfected cells, it did not accumulate to detectable levels in HSV-1-infected cells under identical exposure conditions. Significantly, resistance of translation to the acute ER stress observed in infected cells requires HSV-1 gene expression. Whereas blocking entry into the true late phase of the viral developmental program does not abrogate ER stress-resistant translation, the presence of viral immediate-early proteins is sufficient to establish a state permissive of continued polypeptide synthesis in the presence of ER stress-inducing agents. Thus, one or more previously uncharacterized viral functions exist to counteract the accumulation of phosphorylated eIF2alpha in response to ER stress in HSV-1-infected cells.

Animals↗

PubMed1980

Location of the initiation site for protein synthesis on foot-and-mouth disease virus RNA by in vitro translation of defined fragments of the RNA.

An mRNA-dependent reticulocyte lysate has been used to translate foot-and-mouth disease virus RNA in vitro. Polypeptides P16, P20a, and P88, which have been shown to be derived from the 5' end of the RNA by pactamycin mapping experiments with infected cells, were preferentially synthesized in vitro. Removal of VPg, the small protein covalently linked to the 5' end of the genome RNA, had no effect on the translation of the RNA. The two RNA fragments (L and S) produced by specific digestion of the polycytidylic acid [poly(C)] tract with RNase H were also translated in vitro. The L fragment, consisting of RNA to the 3' side of the poly(C) tract and including the polyadenylic acid [poly(A)] tract, directed the synthesis of the same products as those made by full-length RNA. However, no small defined products were produced when the S fragment, which contains the 5' end of the RNA, was translated. These results show that the major initiation site for protein synthesis on foot-and-mouth disease virus RNA is to the 3' side of the poly(C) tract. Furthermore, the use of N-formyl [35S]methionine tRNAfMet as a label for the initiation peptides showed that the major polypeptide labeled in lysates primed with both full-length RNA and the L fragment was P16, i.e., the protein nearest the initiation site for translation as deduced from pactamycin mapping experiments. Fragments of RNA were also translated in vitro. Those containing the poly(C) tract gave products similar to those produced when full-length RNA was translated. The polypeptides synthesized when fragments containing the poly(A) tract were used, however, did not resemble those made from full-length RNA.

Aphthovirus↗

PubMed1983

Processing of herpes simplex virus proteins and evidence that translation of thymidine kinase mRNA is initiated at three separate AUG codons.

The role which post-translational modification plays in the genesis of herpes simplex virus-induced polypeptides was investigated. Two-dimensional gel electrophoresis was used to identify those polypeptides (i) synthesized in vitro, (ii) labeled in vivo during a pulse, and (iii) labeled after a chase. Excluding glycoproteins, we detected 36 precursor or short-lived polypeptides, 8 polypeptides which were generated by post-translational modification, 46 polypeptides which were apparently not modified after synthesis, and 19 polypeptides which were either transient intermediates or not modified. Comparison of polypeptides synthesized in vitro and during an in vivo pulse showed that translation in vitro resembles quite closely translation in vivo and that amounts of protein synthesized in vivo are determined largely by the levels of mRNA. This analysis provided the basis for an investigation of the suggestion (C.M. Preston and D.J. McGeoch, J. Virol. 38:593-605, 1981) that the two polypeptides of apparent molecular weights of 43,000 (VI 43) and 39,000 (VI 39) encoded by the herpes simplex virus type 1 thymidine kinase gene are translated from a single mRNA by two in-phase initiation codons. Hybrid arrest was used to identify in vitro translation products encoded by the thymidine kinase gene. Two-dimensional gel electrophoresis showed that VI 39 was more acidic than VI 43, consistent with the predicted amino acid composition of a polypeptide whose synthesis was initiated at the second AUG codon, located 135 bases downstream from the first. Furthermore, two-dimensional gels revealed a third polypeptide whose synthesis was arrested by the same fragment. Its pI and apparent molecular weight (38,000) were compatible with initiation of translation at a third AUG codon an additional 42 bases downstream. Our findings provide strong evidence that downstream initiation codons within the thymidine kinase mRNA are used.

Codon↗

PubMed1992

Cap-independent translation by the 5' untranslated region of Theiler's murine encephalomyelitis virus.

The RNA genome of Theiler's murine encephalomyelitis viruses, a picornavirus belonging to the genus Cardiovirus, is translated in infected cells to a polyprotein. Unlike cellular messages, the 5' end of the RNA is not capped, and the untranslated region (UTR) is quite long (1,064 nucleotides in size). In poliovirus and encephalomyocarditis virus, the 5'UTR is thought to mediate cap-independent translation. We report here experiments to determine the role of the Theiler's murine encephalomyelitis virus 5'UTR in translation. Recombinant DNAs were constructed that were transcribed into bicistronic mRNAs encoding 5' chloramphenicol acetyltransferase intercistronic sequences linked to luciferase and a poly(A) 3' tail. The sequences of the 5'UTR, either complete or with sequential 5' deletions, were inserted into the intercistronic region. Bicistronic RNA transcripts were translated in a rabbit reticulocyte lysate or used to transfect BHK-21 cells, and chloramphenicol acetyltransferase and luciferase synthesis was quantitated. The results strongly suggest that the Theiler's virus 5'UTR promotes cap-independent translation and that the 5' boundary of the relevant signals resides 3' to nucleotide 500. Monocistronic mRNAs were synthesized by using an expression vector in which the 5'UTR containing deletions at the 3' terminus was inserted 5' to the coding sequences for luciferase. Analysis of luciferase translation in a rabbit reticulocyte lysate suggests that the 3' end of the translation initiation signal lies between nucleotides 1043 and 1053.

Animals↗

PubMed1993

Translation of the hepatitis B virus P gene by ribosomal scanning as an alternative to internal initiation.

The hepatitis B virus (HBV) P gene which encodes the reverse transcriptase and other proteins required for replication is expressed on the bicistronic mRNA pregenome which also encodes the capsid protein in its first cistron. Recent results have suggested that the hepadnaviral P gene is translated by internal entry of ribosomes upstream from the P gene, in the overlapping C gene. Using a reporter gene fused to the HBV C or P gene, we demonstrate that the C sequence does not allow internal initiation of translation. Alternatively, our results support a model in which the HBV P gene is translated by ribosomes which scan from the capped extremity of the bicistronic mRNA pregenome. The mechanism by which the ribosomes scan past four AUGs before they initiate translation at the P AUG was analyzed. Our results show that these AUGs are skipped via two mechanisms: leaky scanning on AUGs in a weak or suboptimal initiation context and translation of an out-of-C-frame minicistron followed by reinitiation at P AUG. The minicistron translation allows ribosomes to bypass an AUG in a favorable context that would otherwise be used as a start codon for translation of a truncated capsid protein. Our results suggest that this elaborated scanning mechanism permits the coordinate expression of the HBV C and P genes on the viral bicistronic mRNA pregenome.

Base Sequence↗

PubMed1999

Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro.

AUG-unrelated translation initiation was found in an insect picorna-like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3'-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3' boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5' terminus of the capsid-coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5' part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.

Amino Acid Sequence↗