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Vestibular perception and action employ qualitatively different mechanisms. I. Frequency response of VOR and perceptual responses during Translation and Tilt.

To investigate the neural mechanisms that humans use to process the ambiguous force measured by the otolith organs, we measured vestibuloocular reflexes (VORs) and perceptions of tilt and translation. One primary goal was to determine if the same, or different, mechanisms contribute to vestibular perception and action. We used motion paradigms that provided identical sinusoidal inter-aural otolith cues across a broad frequency range. We accomplished this by sinusoidally tilting (20 degrees, 0.005-0.7 Hz) subjects in roll about an earth-horizontal, head-centered, rotation axis ("Tilt") or sinusoidally accelerating (3.3 m/s2, 0.005-0.7 Hz) subjects along their inter-aural axis ("Translation"). While identical inter-aural otolith cues were provided by these motion paradigms, the canal cues were substantially different because roll rotations were present during Tilt but not during Translation. We found that perception was dependent on canal cues because the reported perceptions of both roll tilt and inter-aural translation were substantially different during Translation and Tilt. These findings match internal model predictions that rotational cues from the canals influence the neural processing of otolith cues. We also found horizontal translational VORs at frequencies >0.2 Hz during both Translation and Tilt. These responses were dependent on otolith cues and match simple filtering predictions that translational VORs include contributions via simple high-pass filtering of otolith cues. More generally, these findings demonstrate that internal models govern human vestibular "perception" across a broad range of frequencies and that simple high-pass filters contribute to human horizontal translational VORs ("action") at frequencies above approximately 0.2 Hz.

Adult↗

Three-dimensional organization of otolith-ocular reflexes in rhesus monkeys. III. Responses To translation.

The three-dimensional (3-D) properties of the translational vestibulo-ocular reflexes (translational VORs) during lateral and fore-aft oscillations in complete darkness were studied in rhesus monkeys at frequencies between 0.16 and 25 Hz. In addition, constant velocity off-vertical axis rotations extended the frequency range to 0.02 Hz. During lateral motion, horizontal responses were in phase with linear velocity in the frequency range of 2-10 Hz. At both lower and higher frequencies, phase lags were introduced. Torsional response phase changed more than 180 degrees in the tested frequency range such that torsional eye movements, which could be regarded as compensatory to "an apparent roll tilt" at the lowest frequencies, became anticompensatory at all frequencies above approximately 1 Hz. These results suggest two functionally different frequency bandwidths for the translational VORs. In the low-frequency spectrum (<<0.5 Hz), horizontal responses compensatory to translation are small and high-pass-filtered whereas torsional response sensitivity is relatively frequency independent. At higher frequencies however, both horizontal and torsional response sensitivity and phase exhibit a similar frequency dependence, suggesting a common role during head translation. During up-down motion, vertical responses were in phase with translational velocity at 3-5 Hz but phase leads progressively increased for lower frequencies (>90 degrees at frequencies <0.2 Hz). No consistent dependence on static head orientation was observed for the vertical response components during up-down motion and the horizontal and torsional response components during lateral translation. The frequency response characteristics of the translational VORs were fitted by "periphery/brain stem" functions that related the linear acceleration input, transduced by primary otolith afferents, to the velocity signals providing the input to the velocity-to-position neural integrator and the oculomotor plant. The lowest-order, best-fit periphery/brain stem model that approximated the frequency dependence of the data consisted of a second order transfer function with two alternating poles (at 0.4 and 7.2 Hz) and zeros (at 0.035 and 3.4 Hz). In addition to clearly differentiator dynamics at low frequencies (less than approximately 0.5 Hz), there was no frequency bandwidth where the periphery/brain stem function could be approximated by an integrator, as previously suggested. In this scheme, the oculomotor plant dynamics are assumed to perform the necessary high-frequency integration as required by the reflex. The detailed frequency dependence of the data could only be precisely described by higher order functions with nonminimum phase characteristics that preclude simple filtering of afferent inputs and might be suggestive of distributed spatiotemporal processing of otolith signals in the translational VORs.

Animals↗

Responses of neurons in the nucleus of the basal optic root to translational and rotational flowfields.

The nucleus of the basal optic root (nBOR) receives direct input from the contralateral retina and is the first step in a pathway dedicated to the analysis of optic flowfields resulting from self-motion. Previous studies have shown that most nBOR neurons exhibit direction selectivity in response to large-field stimuli moving in the contralateral hemifield, but a subpopulation of nBOR neurons has binocular receptive fields. In this study, the activity of binocular nBOR neurons was recorded in anesthetized pigeons in response to panoramic translational and rotational optic flow. Translational optic flow was produced by the "translator" projector described in the companion paper, and rotational optic flow was produced by a "planetarium projector" described by Wylie and Frost. The axis of rotation or translation could be positioned to any orientation in three-dimensional space. We recorded from 37 cells, most of which exhibited a strong contralateral dominance. Most of these cells were located in the caudal and dorsal aspects of the nBOR complex and many were localized to the subnucleus nBOR dorsalis. Other units were located outside the boundaries of the nBOR complex in the adjacent area ventralis of Tsai or mesencephalic reticular formation. Six cells responded best to rotational flowfields, whereas 31 responded best to translational flowfields. Of the rotation cells, three preferred rotation about the vertical axis and three preferred horizontal axes. Of the translation cells, 3 responded best to a flowfield simulating downward translation of the bird along a vertical axis, whereas the remaining 28 responded best to flowfields resulting from translation along axes in the horizontal plane. Seventeen of these cells preferred a flowfield resulting from the animal translating backward along an axis oriented approximately 45 degrees to the midline, but the best axes of the remaining eleven cells were distributed throughout the horizontal plane with no definitive clustering. These data are compared with the responses of vestibulocerebellar Purkinje cells.

Animals↗

Barthel index in a Middle-East country: translation, validity and reliability.

BACKGROUND: In Iran, a Middle-East country, no disability scale has been translated and validated for use in stroke clinical trials. This study was designed to translate the Barthel Index and make its Persian translated form valid and reliable. METHODS: All items of the Barthel Index were translated into Persian. Also, the Modified Rankin Scale (MRS) was also translated to Persian. Telephone interview was used as the method of data acquisition. Two interviewers were chosen for this means in order to accelerate data gathering and measure interrater agreement. Samples were selected from Isfahan Cardiovascular Research Center's Stroke Registry Unit, a WHO collaborating center in the center of Iran. All the patients were registered as stroke or intracerebral hemorrhage (ICH). These patients were inhabitants of Isfahan Province who had suffered from stroke or ICH between 12 and 24 months before data acquisition. Chronbach's alpha, test-retest reliability, concurrent validity with MRS, interrater agreement and item analysis were done for the translated questionnaire. RESULTS: Translated questionnaires were filled by interview from 459 stroke patients. Their mean age was 68.11 +/- 11.59 years. 243 of them were male (52.9%). Cronbach's alpha was 0.9354. Spearman's correlation coefficient between translated Barthel Index scores and MRS scores was -0.912. Spearman's correlation coefficient between 2 scores, to determine test-retest reliability was 0.989. Concordance correlation to determine interrater agreement was 0.994. All corrected item-total correlations were greater than 0.5. CONCLUSIONS: The Persian translated version of the Barthel Index is a reliable and valid questionnaire for use in stroke clinical trials.

Activities of Daily Living↗

Quantification of anterior translation of the humeral head in the throwing shoulder. Manual assessment versus stress radiography.

Clinical evaluation of humeral head translation relies mainly on manual tests to measure laxity in the human shoulder. The purposes of this study were to determine whether side-to-side differences exist in anterior humeral head translation in professional baseball pitchers, to compare manual laxity testing with stress radiography for quantifying humeral head translation, and to test intrarater reliability of the manual humeral head translation and stress radiography tests. Twenty professional baseball pitchers underwent bilateral manual anterior humeral head translation and stress radiographic tests. Stress radiography was performed by imparting a 15-daN anterior load to the shoulder in 90 degrees of abduction with both neutral and 60 degrees of external rotation and recording the glenohumeral joint translation at rest and under stress in each position. Eight subjects were retested to assess the reliability of these methods. Results showed no significant difference between the dominant and nondominant extremity in the amount of anterior humeral head translation measured manually and with stress radiography, nor significant correlation between anterior humeral head translation measured manually and by stress radiography. Test-retest reliability was moderate-to-poor for the manual humeral head translation test and moderate for stress radiography.

Adolescent↗

A conserved 90 nucleotide element mediates translational repression of nanos RNA.

Correct formation of the Drosophila body plan requires restriction of nanos activity to the posterior of the embryo. Spatial regulation of nanos is achieved by a combination of RNA localization and localization-dependent translation such that only posteriorly localized nanos RNA is translated. Cis-acting sequences that mediate both RNA localization and translational regulation lie within the nanos 3' untranslated region. We have identified a discrete translational control element within the nanos 3' untranslated region that acts independently of the localization signal to mediate translational repression of unlocalized nanos RNA. Both the translational regulatory function of the nanos 3'UTR and the sequence of the translational control element are conserved between D. melanogaster and D. virilis. Furthermore, we show that the RNA helicase Vasa, which is required for nanos RNA localization, also plays a critical role in promoting nanos translation. Our results specifically exclude models for translational regulation of nanos that rely on changes in polyadenylation.

Animals↗

Differential usage of two in-frame translational start codons regulates subcellular localization of Arabidopsis thaliana THI1.

Arabidopsis thaliana THI1 is encoded by a single nuclear gene and directed simultaneously to mitochondria and chloroplasts from a single major transcript. In vitro transcription/translation experiments revealed the presence of two translational products by the differential usage of two in-frame translational start codons. The coupling site-specific mutations on the THI1 encoding sequence with green fluorescent protein (GFP) gene fusions showed that translation initiation at the first AUG directs translocation of THI1 to chloroplasts. However, when translation starts from the second AUG, THI1 is addressed to mitochondria. Analysis of the translation efficiency of thi1 mRNA revealed that the best context for translation initiation is to use the first AUG. In addition, a suboptimal context in the vicinity of the second AUG initiation codon, next to a stable stem-and-loop structure that is likely to slow translation, has been noted. The fact that translation preferentially occurs in the first AUG of this protein suggests a high requirement for TH1 in chloroplasts. Although the frequency of upstream AUG translation is higher, according to the first AUG rule, initiation at the second AUG deviates significantly from Kozak's consensus. It suggests leaky ribosomal scanning, reinitiation or the internal entry of ribosomes to assure mitochondrial protein import.

Arabidopsis↗

Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES.

The cellular protein, poly(rC) binding protein 2 (PCBP2), is known to function in picornavirus cap-independent translation. We have further examined the RNA binding properties and protein-protein interactions of PCBP2 necessary for translation. We have studied its putative multimerization properties utilizing the yeast two-hybrid assay and in vitro biochemical methods, including glutathione S-transferase (GST) pull-down assays and gel filtration. Through genetic analysis, the multimerization domain has been localized to the second K-homologous (KH) RNA binding domain of the protein between amino acids 125 and 158. To examine the function of multimerization in poliovirus translation, we utilized the truncated protein, DeltaKH1-PCBP2, which is capable of multimer formation, but does not bind poliovirus stem-loop IV RNA (an interaction required for translation). Utilizing RNA binding and in vitro translation assays, this protein was shown to act as a dominant negative, suggesting that PCBP2 multimerization functions in poliovirus translation and RNA binding. Additionally, PCBP2 containing a deletion in the multimerization domain (DeltaKH2-PCBP2) was not able to bind poliovirus stem-loop IV RNA and could not rescue translation in extracts that were depleted of endogenous PCBP2. Results from these experiments suggest that the multimerization of PCBP2 is required for efficient RNA binding and cap-independent translation of poliovirus RNA. By examining the functional interactions of the cellular protein PCBP2, we have discovered a novel determinant in the mechanism of picornavirus cap-independent translation.

DNA-Binding Proteins↗

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↗

Direct binding of thyrotropin receptor autoantibody to in vitro translated thyrotropin receptor: a comparison to radioreceptor assay and thyroid stimulating bioassay.

Graves' disease is characterized by the presence of autoantibodies to the thyrotropin receptor (TSHR), which are pathogenic and responsible for disease activity. It is well recognized that the autoantibodies are heterogeneous and recognize a number of different conformational dependent epitopes on the TSHR. In this study, we have extended our previous observations to study the interaction of Graves' disease autoantibodies with TSHR ectodomain produced by in vitro transcription and translation reaction. The specific activity of the translated TSHR ectodomain has been increased by a log fold by adding an efficient ribosome binding Kozak sequence before the translation initiation codon as well as double labelling with 35S-methionine and 35S-cysteine during the translation reaction. Addition of canine pancreatic microsomes to the translation mix showed that the glycosylation of TSHR ectodomain did not occur efficiently for the nascent receptor protein. In order to determine the specificity and sensitivity of the improved assay with nonglycosylated TSHR ectodomain, we have studied 331 sera from Graves' disease patients and as controls 100 sera from patients with nonthyroid autoimmune disorders as well as sera from 200 normal control subjects with no family history of thyroid autoimmunity. With this large cohort of sera from Graves' disease and control individuals, 25% of Graves' disease sera immunoprecipitated the dual labeled, in vitro transcribed and translated TSHR ectodomain, exceeding the 98th percentile of the control sera. There was no correlation between the autoantibodies that immunoprecipitate the in vitro translated TSHR ectodomain and those that inhibit iodinated TSH binding in the radioreceptor assay and those with biological activity in a bioassay. The data are consistent with the finding that a proportion of Graves' disease autoantibodies can interact directly with TSHR ectodomain produced by in vitro transcription and translation. However, in contrast to the wide use of similar translation and immunoprecipitation assays to measure other autoantibodies for the diagnosis of autoimmune disorders, such as type 1 diabetes, the TSHR immunoprecipitation on its own is unsuitable for diagnosis of Graves' disease.

Animals↗

Definition, quantification, and correction of translation deformities using long leg, frontal plane radiography.

The surgical realignment of mechanical axis deviation is necessary to prevent early joint degeneration. Modern types of external fixation systems allow alignment of the mechanical axis to exact degrees. Predominately, these are corrections of angulation deformities. In some cases, the analysis of the mechanical axis deviation does not show any angulation deformity, but rather a parallel staggering of the mechanical axis lines of a bone. Such parallel staggering of the mechanical axis lines is defined as a translation deformity of the bone. In combined deformities with angulation and translation, the center of deformity can be established proximal or distal to the limit of the bone. In translation deformities, the realignment of the mechanical axis requires a parallel restaggering made by a translation-osteotomy or by a counterangulated double osteotomy. In complex deformities with angulation and translation, the translation requires separate corrective planning. In frontal plane radiographs of the standing leg, the components of angulation and translation can be established graphically or by simple trigonometric formulas. The analysis and surgical procedure to realign translation deformities or a combination of translation and angulation deformities using an unilateral fixator device are discussed.

Adult↗

Positive and negative effects on translation of the hepatitis C virus 3' untranslated region.

OBJECTIVES: To determine the role of the hepatitis C virus 3' untranslated region in viral mRNA translation in transfected cells and in cell extracts. STUDY DESIGN/METHODS: Noninfectious hepatitis C virus mini-genome RNAs with various deletions in the viral 3' untranslated region were transfected into cells or translated in vitro, and the translation efficiency was determined. RESULTS: We have found that the presence of the hepatitis C virus 3' untranslated region modestly increases mRNA translation. The positive effect correlated with the binding of a 45-kDa cytoplasmic factor to the hepatitis C virus 3' untranslated region. Furthermore, the U-rich sequence in the hepatitis C virus 3' untranslated region inhibits translation of capped and polyadenylated mRNAs as a result of the hybridization. CONCLUSIONS: The modest effect of the hepatitis C virus 3' untranslated region on translation suggests that it does not play a major role in mRNA translation. The inhibitory effect of the hepatitis C virus 3' untranslated region on translation of polyadenylated mRNAs supports the notion that translation of hepatitis C virus mRNAs occurs independently of a polyA tail.

3' Untranslated Regions↗

The absence of a m7G cap on beta-globin mRNA and alfalfa mosaic virus RNA 4 increases the amounts of initiation factor 4F required for translation.

beta-Globin mRNA and alfalfa mosaic virus (AMV) RNA 4, two naturally capped mRNAs, and satellite tobacco necrosis virus (STNV) RNA, a naturally uncapped mRNA, were prepared by in vitro transcription with and without a 5' m7G cap structure (m7G(5')ppp(5')N). The translation of the capped and uncapped forms of these mRNAs was measured in a crude S30 system and a partially purified system from wheat germ. In the S30 system the uncapped forms of beta-globin mRNA and AMV RNA 4 are much less active (greater than or equal to 10%) than their capped forms, whereas the uncapped and capped forms of STNV RNA are equally active. The low activity of uncapped beta-globin mRNA and AMV RNA 4 in the S30 system is due, in part, to inactivation of the uncapped mRNAs in this system. Additional studies, carried out in the partially purified system in which very little inactivation of the mRNAs occurs, show that the uncapped and capped forms of beta-globin mRNA or AMV RNA 4 differ markedly with respect to the amount of eukaryotic initiation factor (eIF)-4F required for translation. For beta-globin mRNA the absence of the 5' cap structure increases the concentration of eIF-4F required for half-maximal translation about 6-fold (from 10 to 60 nM) and for AMV RNA 4 it increases the concentration of eIF-4F about 12-fold (from 5 to 60 nM). The concentrations of eIF-3, eIF-4A, and eIF-4B required for half-maximal translation of the uncapped forms of beta-globin mRNA and AMV RNA 4 are either the same or only slightly higher (1.5- to 2-fold) than the concentrations required for the capped forms. With STNV RNA the concentration of eIF-4F required for half-maximal translation of either uncapped or capped STNV RNA is 3 nM, and the concentrations of eIF-3, eIF-4A, and eIF-4B required for the two forms are also the same. The translation of the capped and uncapped forms of beta-globin mRNA and AMV RNA 4 is inhibited strongly by low concentrations of m7GTP in the partially purified system containing low concentrations of eIF-4F. Under the same conditions, the translation of capped or uncapped STNV RNA is inhibited only slightly by m7GTP. These findings suggest the possibility that the mechanism by which eIF-4F interacts and initiates translation with naturally uncapped mRNAs may not be identical to the mechanism by which eIF-4F interacts and initiates translation of naturally capped mRNAs.

Animals↗

The mechanism of translational coupling in Escherichia coli. Higher order structure in the atpHA mRNA acts as a conformational switch regulating the access of de novo initiating ribosomes.

Bacterial genes are commonly transcribed to form polycistronic mRNAs bearing reading frames whose respective translational efficiencies are not independently determined. As in many bacterial operons, expression of the atp genes of Escherichia coli is strongly influenced by translational coupling. The gene pair atpHA is tightly coupled, whereby atpA is translated at least three times more efficiently than atpH. However, there is no fixed stoichiometry of coupling: mutations in atpH lead to increases in the translation ratio (atpA/atpH) of up to approximately 40-fold. We have demonstrated that secondary structure sequestering the atpA translational initiation region (TIR) is important to the coupling mechanism in that it inhibits de novo translational initiation at the atpA start codon. Genetic and structural analyses indicate that this inhibitory structure can be induced to refold into a less inhibitory conformation either by introducing two single-base substitutions or as a result of ribosomes translating atpH. We propose a model in which the secondary structure of the atpA TIR acts analogously to a "gating device" in that it restricts de novo ribosomal initiation until it is "switched" into a more open conformation. This contrasts with the function of a stem-loop structure located immediately downstream of atpA and upstream of the Shine-Dalgarno region of atpG, which was found to inhibit translation, but not to mediate tight coupling. Results obtained using the "specialized" ribosome system of Hui and de Boer ((1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4762-4766) indicate that primarily ribosomes reinitiating after termination on atpH are responsible for inducing refolding of the atpA TIR. The principle of alternative mRNA conformations with different functional properties embodied in the model presented here can only be fulfilled by certain types of structure. It is likely to operate in several steps of prokaryotic gene expression, underlying a range of regulatory events including transcriptional attenuation and translational activation.

Base Sequence↗

Inhibition of translational initiation in the yeast Saccharomyces cerevisiae as a function of the stability and position of hairpin structures in the mRNA leader.

A new modular in vivo/in vitro expression system was constructed which facilitates studies of the control and regulation of gene expression in the yeast Saccharomyces cerevisiae. We studied the influence of stem-loop structures inserted into the non-translated leader region upon the steady-state levels and translation of mRNAs bearing the cat gene from the bacterial transposon Tn9. mRNA abundance changed relatively little in response to alterations in the leader sequence and structure, whereas stem-loop structures clearly inhibited translation to a degree that was dependent upon the predicted stability as well as the position of the inserted secondary structure. A stem-loop structure with a predicted stability greater than -28 kcal mol-1 and with a stem comprising at least 15 (mainly G/C) base pairs inhibited translation in vivo by at least 98%. A stem-loop structure with a predicted stability of approximately -14 kcal mol-1, whose stem comprised at least six G/C base pairs, inhibited translation in vivo by at least 66%. The hairpins were more inhibitory when placed close to the start codon than when positioned near the 5' end of the leader. An mRNA showing extensive complementarity between the leader and trailer regions was not only poorly translated but also had a steady-state level at least three times higher than the average for all the cat constructs examined. Translation of the various mRNAs in a yeast cell-free system followed qualitatively the same pattern as the results obtained in vivo. The stem-loop structures were far less inhibitory in a reticulocyte lysate system. Overall, the data are likely to reflect the full spectrum of translational activities of yeast mRNAs in vivo determined by secondary structure and emphasize the importance of translation as a control step in gene expression.

Animals↗

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

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

Animals↗

Regulated poly(A) tail shortening in somatic cells mediated by cap-proximal translational repressor proteins and ribosome association.

The poly(A) tail plays an important role in translation initiation. We report the identification of a mechanism that operates in mammalian somatic cells, and couples mRNA poly(A) tail length with its translation state. The regulation of human ferritin L-chain mRNA by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) is subject to this mechanism: translational repression imposed by IRP binding to the IRE of ferritin L-chain mRNA induces poly(A) tail shortening. For the accumulation of mRNAs with short poly(A) tails, IRP binding to an IRE per se is not sufficient, but must cause translational repression. Interestingly, puromycin and verrucarin (general translation inhibitors that dissociate mRNAs from ribosomes) mimick the negative effect of the specific translational repressor proteins on poly(A) tail length, whereas cycloheximide and anisomycin (general translation inhibitors that maintain the association between mRNAs and ribosomes) preserve long poly(A) tails. Thus, the ribosome association of the mRNA appears to represent the critical determinant. These findings identify a novel mechanism of regulated polyadenylation as a consequence of translational control. They reveal differences in poly(A) tail metabolism between polysomal and mRNP-associated mRNAs. A possible role of this mechanism in the maintenance of translational repression is discussed.

Animals↗

Inhibition of HeLa cell messenger RNA translation by 7-methylguanosine 5'-monophosphate.

Translation of HeLa cell RNA containing poly(A) in a wheat germ cell-free system is markedly but incompletely inhibited by 7-methylguanosine 5'-monophosphate (m7G5'p). We have analyzed the translation products synthesized in the presence of different concentrations of m7G5'p and find that translation of all mRNAs is equally inhibited. To demonstrate the specificity of the inhibitor for RNAs with 5'-terminal m7G5' ppp... we show that specific translation products of satellite tobacco necrosis virus RNA, which does not have this 5' terminus, are synthesized in the presence of m7G5' p. Protein synthesis programmed by endogenous mRNA in a HeLa cell-free system is inhibited after a 10-min lag by m7G5' p. Other guanosine nucleotides without the 7-methyl group or with the phosphate in a different position are not inhibitor. We show that translation of all mRNAs is inhibited to a similar extent by m7G5'p in the HeLa cell-free system, by synthesizing 35S-labeled proteins in the presence of different inhibitory concentrations of this nucleotide and analyzing the translation products by electrophoresis and autoradiography. Translation of encephalomyocarditis virus RNA added to the HeLa cell-free system is not inhibited by m7"g5p; this viral RNA does not have this nucleotide at the 5' terminus. This indicates that m7G5'p specifically inhibits translation of mRNAs with the 5' terminus m7G5'ppp... and suggests that initiation of translation of picornavirus RNA may proceed via a mechanism different from that of cellular mRNAs.

Globins↗