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PubMed1997

Poly(A) dependent translation in rabbit reticulocyte lysate.

Poly(A) tail has been known to enhance mRNA translation in eukaryotic cells. However, the effect of poly(A) tail in in vitro is rather small. Rabbit reticulocyte lysate (RRL) is widely used for studying translation in vitro. Translation in RRL is typically performed in nuclease-treated lysate in which most of the endogenous mRNA have been removed. In this condition, the difference in the translational efficiency between poly(A)+ and poly(A)- mRNAs is about two-fold. We studied the effect of poly(A) tail on luciferase mRNA translation in nuclease untreated rabbit reticulocyte lysate, in which endogenous globin mRNAs were actively translated. In the case of capped mRNAs, stimulation of translation by poly(A) addition was about 1.5- to 1.6-fold and the effect of the poly(A) length was small. However, in the case of uncapped mRNAs, the addition of poly(A) tail increased luciferase expression over 10-fold. The effect of the poly(A) tail was dependent on its length. The difference in the translational efficiency was not due to the change of mRNA stability. These data indicate that RRL has the potential to translate mRNA in a poly(A) dependent manner.

Animals↗

PubMed2000

Translation from the internal ribosome entry site of bovine viral diarrhea virus is independent of the interaction with polypyrimidine tract-binding protein.

Translation of the pestiviral polyprotein is initiated cap independently at an internal site of the viral RNA, the internal ribosome entry site (IRES). We investigated the translation from the IRES of bovine viral diarrhea virus (BVDV) and the possible interaction of the unconventional cellular RNA-binding proteins, particularly of polypyrimidine tract-binding protein (PTB). The BVDV IRES is translationally active in rabbit reticulocyte lysate (RRL), and it is translated most efficiently at low concentrations of Mg(2+)- and K(+)-ions. In the UV cross-link assay, several proteins from RRL bind to the BVDV IRES, including proteins of 50, 65 and 72kDa, but no protein of 57kDa possibly corresponding to PTB, although PTB is endogenously present in RRL. However, the BVDV IRES can bind PTB weakly under certain conditions. Interestingly, in a functional depletion and add-back translation system, PTB does not enhance translation of BVDV, although PTB enhances translation of a picornavirus in this translation stimulation assay. These results indicate that PTB can bind the BVDV IRES RNA, but translation is independent of the action of PTB.

Animals↗

PubMedDate not supplied

Effect of humeral head component size on hemiarthroplasty translations and rotations.

Glenohumeral translation and rotation were measured in 6 grossly normal, fresh frozen shoulder preparations while a manual load was applied to the humerus. The same tests (maximum elevation, total rotation, anterior/posterior (A/P) translation, and inferior translation) were repeated for each shoulder through 8 series: 1 with the shoulder intact, 1 with the shoulder vented, and 6 with progressively larger humeral head components after hemiarthroplasty. There was an inverse linear relation between humeral head component size and all 4 outcome variables. Replacing the native head with a component of equal diameter reduced elevation 20%, rotation 40%, A/P translation 50%, and inferior translation 60% in the vented shoulder. Replacing the native head with a component of equal effective volume decreased elevation 8%, rotation 20%, A/P translation 25%, and inferior translation 40% in the vented shoulder. Increasing humeral head component size decreased rotation, A/P translation, and inferior translation by similar percentages and elevation somewhat less. Humeral head component size is better described in terms of volume than in terms of diameter or offset.

Arthroplasty, Replacement↗

PubMed1998

Translational activation of uncapped mRNAs by the central part of human eIF4G is 5' end-dependent.

Translation initiation factor (eIF) 4G represents a critical link between mRNAs and 40S ribosomal subunits during translation initiation. It interacts directly with the cap-binding protein eIF4E through its N-terminal part, and binds eIF3 and eIF4A through the central and C-terminal region. We expressed and purified recombinant variants of human eIF4G lacking the N-terminal domain as GST-fusion proteins, and studied their function in cell-free translation reactions. Both eIF4G lacking its N-terminal part (aa 486-1404) and the central part alone (aa 486-935) exert a dominant negative effect on the translation of capped mRNAs. Furthermore, these polypeptides potently stimulate the translation of uncapped mRNAs. Although this stimulation is cap-independent, it is shown to be dependent on the accessibility of the mRNA 5' end. These results reveal two unexpected features of eIF4G-mediated translation. First, the C-terminal eIF4A binding site is dispensable for activation of uncapped mRNA translation. Second, translation of uncapped mRNA still requires 5' end-dependent ribosome binding. These new findings are incorporated into existing models of mammalian translation initiation.

Escherichia coli↗

PubMed1987

Opposite responses of rabbit and human globin mRNAs to translational inhibition by cap analogues.

The translational efficiency of an mRNA may be determined at the step of translational initiation by the efficiency of its interaction with the cap binding protein complex. To further investigate the role of these interactions in translational control, we compare in vitro the relative sensitivities of rabbit and human alpha- and beta-globin mRNAs to translational inhibition by cap analogues. We find that rabbit beta-globin mRNA is more resistant to translational inhibition by cap analogues than rabbit alpha-globin mRNA, while in contrast, human beta-globin mRNA is more sensitive to cap analogue inhibition than human alpha-globin mRNA. This opposite pattern of translational inhibition by cap analogues of the rabbit and human alpha- and beta-globin mRNAs is unexpected as direct in vivo and in vitro comparisons of polysome profiles reveal parallel translational handling of the alpha- and beta-globin mRNAs from these two species. This discordance between the relative translational sensitivities of these mRNAs to cap analogues and their relative ribosome loading activities suggests that cap-dependent events may not be rate limiting in steady-state globin translation.

Animals↗

PubMed1998

Dual function of the messenger RNA cap structure in poly(A)-tail-promoted translation in yeast.

The messenger RNA 3' poly(A) tail critically affects the initiation and control of translation in eukaryotes. By analogy to elements involved in transcription initiation, the poly(A) tail has been described as a 'translational enhancer' that enhances the 'translational promoter' activity of the mRNA 5'-cap structure. Elongation or shortening of the poly(A) tail regulates translation during development. Here we show, using cell-free and in vivo translation analyses in Saccharomyces cerevisiae, that the poly(A) tail can act as an independent 'translational promoter', delivering ribosomes to uncapped mRNAs even if their 5' end is blocked. When mRNAs compete for ribosome binding, neither the cap structure nor the poly(A) tail alone is enough to drive efficient translation, but together they synergize and direct ribosome entry to the 5' end. The cap structure both promotes ribosome recruitment, together with the poly(A) tail, and tethers recruited ribosomes to the 5' end. Correct choice of translation initiation codons and the function of translational regulators acting on the 5' untranslated region are thus ensured by the functional interaction of the poly(A) tail with the cap structure.

Base Sequence↗

PubMed1985

Studies of translatable mRNA for rabbit C-reactive protein.

C-reactive protein (CRP) mRNA was assayed by cell-free translation of poly(A)-containing liver RNA isolated both from rabbits stimulated to undergo the acute-phase response and from unstimulated control rabbits. No CRP-related translation products were identified until the denaturant methylmercury hydroxide (CH3HgOH) was added to the RNA before cell-free translation. In the presence of the denaturant, a 24000-Da translation product was synthesized which was immunochemically identifiable as the CRP primary translation product. It is likely that rabbit CRP mRNA can form a stable intramolecular duplex which interferes with its translatability in vitro. The 24000-Da CH3HgOH-facilitated cell-free translation product was not detected in poly(A)-containing liver RNA from unstimulated animals, indicating that the concentration of translatable CRP mRNA was dramatically induced during the acute-phase response. On the basis of absorption experiments, the 24000-Da CRP primary translation product was immunochemically more closely related to denatured CRP than to native CRP.

Animals↗

PubMed1994

Translation initiation on the insulin-like growth factor II leader 1 is developmentally regulated.

The majority of cellular mRNAs have relatively short and unstructured 5' untranslated regions (UTRs) that allow efficient translation, such as the beta-globin mRNA. An exception to this rule is the group of growth factor mRNAs which, in general, have long 5' UTRs with a high G + C content. An example is insulin-like growth factor II (IGF-II), which is encoded by four mRNAs, arising from four different promoters. Transcripts having the human IGF-II leader 1 are only expressed in adult liver where IGF-II protein synthesis is solely under direction of this 5' UTR. We investigated the translational efficiency in vitro of this 5' UTR, linked to the chloramphenicol acetyltransferase (CAT) encoding region. As expected from the primary structure of IGF-II leader 1, translational efficiency was very low compared with beta-globin 5' UTR-CAT mRNA. Addition of cell extract from undifferentiated P19 embryonal carcinoma (EC) cells preferentially stimulated translation of an IGF-II 5' UTR RNA construct. No translational stimulation was found when cell extract from differentiated P19 EC cells was added. In contrast with the beta-globin 5' UTR, translation initiation on the IGF-II 5' UTR was not dependent on the presence of a cap structure. The results imply that only in undifferentiated P19 EC cells and not in their differentiated derivatives is a factor present that specifically stimulates IGF-II RNA translation, thereby suggesting translational regulation of IGF-II production during early embryonic development. A mechanism for translation initiation on the 5' UTR of IGF-II is discussed.

Adenoviridae↗

PubMed1997

Tissue-specific promoters of the alpha human folate receptor gene yield transcripts with divergent 5' leader sequences and different translational efficiencies.

The alpha human folate receptor (alphahFR), or KB cell folate receptor, gene contains two major promoters that produce transcripts, KB1 and KB4, varying only in the length and sequence of their 5' untranslated regions (UTRs). Using RNase protection assays specific for each isoform, we show that the level of expression of these two transcripts is tissue-specific, indicating that promoter usage is regulated, not constitutive. RNA stabilities and translational efficiencies of the KB1 and KB4 transcripts were compared to determine the functional significance of the different 5' UTRs. Analyses of RNA turnover in vivo with actinomycin D to block new transcription and in vitro with a cytoplasmic extract indicate no discernible differences in the stabilities of the two transcripts. However, the KB4 transcript is 2-3-fold more efficiently translated in wheat germ extracts in vitro and transfected CHO cells in vivo. Also, high ionic strength, which favours the formation of RNA secondary structure, differentially affects the translational efficiencies of the two transcripts. Translation of the longer KB1 mRNA is 2-5-fold more inhibited by hypertonic conditions than translation of the KB4 mRNA. Because the 5' UTR of KB1 is approximately four times longer than the 5' UTR of KB4, 149 bp (75%) of the KB1 5' UTR were deleted to determine whether the long leader sequence inhibited translation. The resulting derivative, dKB1, has a 5' UTR similar in length, but not sequence, to the 5' UTR of KB4. dKB1 is translated at a level approaching that of KB4 in wheat germ extracts, indicating that the upstream portion of the 5' leader sequence contributes to the relative translational inefficiency of KB1. Hence, one consequence of tissue-specific promoter usage is the production of alphahFR transcripts with different 5' non-coding regions that affect translational efficiency.

Animals↗

PubMed2007

What's next in translational medicine?

Translational medicine is the integrated application of innovative pharmacology tools, biomarkers, clinical methods, clinical technologies and study designs to improve disease understanding, confidence in human drug targets and increase confidence in drug candidates, understand the therapeutic index in humans, enhance cost-effective decision making in exploratory development and increase phase II success. Translational research is one of the most important activities of translational medicine as it supports predictions about probable drug activities across species and is especially important when compounds with unprecedented drug targets are brought to humans for the first time. Translational research has the potential to deliver many practical benefits for patients and justify the extensive investments placed by the private and public sector in biomedical research. Translational research encompasses a complexity of scientific, financial, ethical, regulatory, legislative and practical hurdles that need to be addressed at several levels to make the process efficient. Several have resisted the idea of supporting translational research because of its high costs and the fear that it may re-direct funds from other biomedical disciplines. Resistance also comes from those more familiar with traditional clinical research methods. In this review, we argue that translational research should be seen as enabled by ongoing efforts in basic and clinical research and not competing with them. Translational research provides the knowledge necessary to draw important conclusions from clinical testing regarding disease and the viability of novel drug mechanisms. Advancing translational research requires education and new sources of funding. This could be achieved through public and congressional education by a joint coalition of patients' advocacy groups, academia, drug regulatory agencies and industry.

Animals↗

PubMed1996

Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo.

In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene (tnp), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3). Several lines of evidence led us to conclude that transposase expression, per se, was not increased. Rather, these mutations appear to increase expression of the tnp'-'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons. To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons. Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These potential start codons fall into distinct classes with regard to translation efficiency in vivo: Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1-3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification, importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3-5-fold, and this same effect was seen in other gene contexts. Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo, consistent with earlier in vitro observations. We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function.

Bacterial Proteins↗

PubMed2002

Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control.

It is commonly believed that the translational efficiency of prokaryotic mRNAs is intrinsically determined by both primary and secondary structures of their translational initiation regions. However, for leaderless mRNAs starting with the AUG initiating codon occurring in bacteria, archaea and eukaryotes, there is no evidence for ribosomal recruitment signals downstream of the 5'-terminal AUG that seems to be the only necessary and constant element. Studies in Escherichia coli have brought to light that the ratio of initiation factors IF2 and IF3 plays a decisive role in translation initiation of leaderless mRNA, indicating that the translational efficiency of this mRNA class can be modulated depending on the availability of components of the translational machinery. Recent data suggested that the start codon of bacterial leaderless mRNAs is recognized by a ribosome-IF2-fMet-tRNA complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes, which points to a conceptual similarity in all initiation pathways. In fact, the faithful translation of leaderless mRNAs in heterologous systems shows that the ability to translate leaderless mRNAs is an evolutionarily conserved function of the translational apparatus.

5' Untranslated Regions↗

PubMed1994

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↗

PubMed2004

Translational regulation of XIAP expression and cell survival during hypoxia in human cholangiocarcinoma.

BACKGROUND & AIMS: Tumor progression is promoted by the ability of tumor cells to resist adverse environmental conditions such as hypoxia. We have shown that translational dysregulation contributes to transformed cell growth in malignant cholangiocytes. Translational regulation of gene expression can contribute to an immediate and rapid response to environmental changes such as hypoxia. Thus, our aims were to assess translational mechanisms involved in cell survival during hypoxia and to identify specific translationally regulated proteins involved in the cellular response to hypoxia. METHODS: Cell viability and apoptosis in response to hypoxia were assessed in human cholangiocarcinoma cells. Translational processes were deregulated by cycloheximide or rapamycin or by targeted deletion of eukaryotic initiation factor (eIF)-4E, a rate-limiting translational initiation factor using small interfering RNA (siRNA). A protein antibody microarray was used to screen for eIF-4E-dependent proteins expressed during hypoxia. Expression of the X-linked inhibitor of apoptosis (XIAP) was decreased using siRNA. RESULTS: Malignant cholangiocytes are resistant to hypoxia-induced apoptosis. Furthermore, cell survival during hypoxia required protein translation. eIF-4E was over expressed in malignant cholangiocytes. Reduction in eIF-4E expression by siRNA decreased tumor cell resistance to hypoxia, increased caspase-3 activation and apoptosis, and decreased cell survival compared with controls. XIAP was identified as a translationally regulated protein expressed during hypoxia. Modulation of XIAP expression by siRNA decreases cell death during hypoxia in vitro and in vivo. CONCLUSIONS: Human cholangiocarcinoma cells are highly resistant to hypoxia. Translational regulation of survival proteins such as XIAP is a mechanism mediating cholangiocarcinoma survival during hypoxia.

Apoptosis↗

PubMedDate not supplied

Glenohumeral translation after arthroscopic thermal capsuloplasty with a radiofrequency probe.

The purpose of this study was to determine whether there are changes in anterior and posterior glenohumeral translation after arthroscopic thermal capsuloplasty with a radiofrequency probe. Anteriorly directed loads of 15 N and 20 N were sequentially applied to the humerus of each of 5 cadaveric glenohumeral joints, and anterior translation on the glenoid was measured through use of a customized translation apparatus and an electromagnetic tracking device. The tests were then repeated with posteriorly directed forces, and posterior translation was measured. During testing, the glenoid was rigidly fixed and the glenohumeral joint was positioned to simulate 90 degrees of shoulder abduction and 90 degrees of external rotation. By means of the radiofrequency probe, thermal energy was then applied to the anteroinferior capsuloligamentous structures; anterior and posterior translation measurements were repeated. The results showed a significant reduction in anterior and posterior translations after thermal capsuloplasty (P < .05). Anterior translation decreased from 6.8 to 4.0 mm (a 41% decrease) with the 15-N load and from 8.6 to 4.9 mm (a 42% decrease) with the 20-N load. Posterior translation decreased from 9.3 to 5.8 mm (a 36% decrease) with the 15-N load and from 10.4 to 6.5 mm (a 35% decrease) with the 20-N load. The results of this study indicate that the radiofrequency probe can be used to decrease both anterior and posterior glenohumeral translation in vitro. The biological effect on heat-treated tissues over time needs to be studied to prove that this is a satisfactory treatment for glenohumeral instability.

Aged↗

PubMedDate not supplied

Ligamentous restraints to anterior and posterior translation of the sternoclavicular joint.

This experiment was conducted to determine the primary ligamentous restraints to anterior and posterior translation of the sternoclavicular joint. Twenty-four unpaired cadaver specimens were mounted in a custom fixture. Anterior and posterior translations were measured under a sub-failure load in the intact specimen and again after transecting one randomly chosen ligament (anterior capsule, posterior capsule, interclavicular ligament, and costoclavicular ligament; n = 6 for each group). Cutting the posterior capsule resulted in significant increases in anterior translation and posterior translation. Cutting the anterior capsule produced significant increases in anterior translation. Cutting the costoclavicular and interclavicular ligaments had little effect on sternoclavicular joint translation. The posterior capsule is the most important restraint for anterior and posterior translation of the sternoclavicular joint. The anterior capsule is another important restraint for anterior translation. The costoclavicular and interclavicular ligaments have little effect on anterior or posterior translation of the sternoclavicular joint.

Aged↗

PubMedDate not supplied

Glenohumeral joint translation after arthroscopic thermal capsuloplasty of the rotator interval.

The treatment of recurrent anterior, posterior, or multidirectional glenohumeral instability by operative closure of the rotator interval has been proposed. The purpose of this study was to determine whether arthroscopic thermal capsuloplasty of the rotator interval with the radiofrequency probe significantly decreases anterior-posterior glenohumeral translation. Anterior and posterior loads of 10, 15, and 20 N were sequentially applied to 8 cadaveric shoulder joints while mounted in a translation testing apparatus with an electromagnetic tracking device measuring anterior and posterior glenohumeral translation. Arthroscopic thermal capsuloplasty was then performed on the rotator interval with a radiofrequency probe. The identical anterior-posterior loading protocol was then repeated, and translations were recorded. The results showed a significant reduction in anterior and posterior translation after thermal capsuloplasty. After rotator interval thermal capsuloplasty, anterior translation decreased by 31.5%, 28.8%, and 27.2% for the 10-, 15-, and 20-N loads, respectively. Posterior translation decreased by 43.1%, 43.8%, and 40.7%, respectively. The results of this study indicate that arthroscopic thermal capsuloplasty of the rotator interval is an effective way by which to decrease both anterior and posterior glenohumeral translation in vitro. To date, no in vivo studies that sufficiently document long-term clinically successful outcomes of such a procedure exist. Future studies are warranted to evaluate the effects of the biologic response to thermal shrinkage and whether these reduced glenohumeral translations will be maintained.

Aged↗

PubMed2002

The caspase-cleaved DAP5 protein supports internal ribosome entry site-mediated translation of death proteins.

Apoptosis is characterized by a translation switch from cap-dependent to internal ribosome entry site (IRES)-mediated protein translation. During apoptosis, several members of the eukaryotic initiation factor (eIF)4G family are cleaved specifically by caspases. Here we investigated which of the caspase-cleaved eIF4G family members could support cap-independent translation through IRES elements that retain activity in the dying cell. We focused on two major fragments arising from the cleavage of eIF4GI and death-associated protein 5 (DAP5) proteins (eIF4GI M-FAG/p76 and DAP5/p86, respectively), because they are the only potential candidates to preserve the minimal scaffold function needed to mediate translation. Transfection-based experiments in cell cultures indicated that expression of DAP5/p86 in cells stimulated protein translation from the IRESs of c-Myc, Apaf-1, DAP5, and XIAP. In contrast, these IRESs were refractory to the ectopically expressed eIF4GI M-FAG/p76. Furthermore, our study provides in vivo evidence that the caspase-mediated removal of the C-terminal tail of DAP5/p97 relieves an inhibitory effect on the protein's ability to support cap-independent translation through the DAP5 IRES. Altogether, the data suggest that DAP5 is a caspase-activated translation factor that mediates translation through a repertoire of IRES elements, supporting the translation of apoptosis-related proteins.

Apoptosis↗