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Oskar allows nanos mRNA translation in Drosophila embryos by preventing its deadenylation by Smaug/CCR4.

Anteroposterior patterning of the Drosophila embryo depends on a gradient of Nanos protein arising from the posterior pole. This gradient results from both nanos mRNA translational repression in the bulk of the embryo and translational activation of nanos mRNA localized at the posterior pole. Two mechanisms of nanos translational repression have been described, at the initiation step and after this step. Here we identify a novel level of nanos translational control. We show that the Smaug protein bound to the nanos 3' UTR recruits the deadenylation complex CCR4-NOT, leading to rapid deadenylation and subsequent decay of nanos mRNA. Inhibition of deadenylation causes stabilization of nanos mRNA, ectopic synthesis of Nanos protein and head defects. Therefore, deadenylation is essential for both translational repression and decay of nanos mRNA. We further propose a mechanism for translational activation at the posterior pole. Translation of nanos mRNA at the posterior pole depends on oskar function. We show that Oskar prevents the rapid deadenylation of nanos mRNA by precluding its binding to Smaug, thus leading to its stabilization and translation. This study provides insights into molecular mechanisms of regulated deadenylation by specific proteins and demonstrates its importance in development.

Animals↗

Translational repression by MSY4 inhibits spermatid differentiation in mice.

In developing male germ cells, newly synthesized protamine mRNAs are stored for up to 7 days before translational activation. Translational repression of protamine 1 (Prm1) mRNA requires sequences present in its 3' untranslated region (UTR) and substantial evidence suggests a role for the murine Y-box protein MSY4 in this process. To determine if MSY4 can mediate translational repression in vivo, we generated transgenic mice in which the temporal window of MSY4 expression was extended during spermatogenesis. Expression of MSY4 disrupted the normal completion of spermatogenesis and caused dominant sterility. Immunocytochemical analysis of several markers, including the protamines, indicated that MSY4 prevented normal activation of translation. mRNAs whose translation was inhibited contained at least one MSY4 RNA recognition site, suggesting sequence-dependent translational repression. Altered translational activation resulted in defective processing of protamine 2 and severe defects in sperm morphogenesis. These results suggest that MSY4 plays an active role in translational repression of several mRNAs in differentiating spermatids.

3' Untranslated Regions↗

Translational augmentation of pro-matrix metalloproteinase 3 (prostromelysin 1) and a tissue inhibitor of metalloproteinases (TIMP)-1 mRNAs by epidermal growth factor and transforming growth factor alpha, but not by interleukin 1 alpha or 12-O-tetradecanoylphorbol 13-acetate in human uterine cervical fibroblasts: the possible involvement of an atypical protein kinase C.

We have previously reported that epidermal growth factor (EGF) augments the translation of pro-matrix metalloproteinase 3 (proMMP-3/prostromelysin 1) and tissue inhibitor of metalloproteinases (TIMP)-1 mRNAs during the first 1-h treatment of human uterine cervical fibroblasts (Hosono, T. et al., FEBS Lett., 381, 115-118, (1996)). In this report, we have investigated the effect of interleukin 1 alpha (IL-1 alpha) and 12-O-tetradecanoylphorbol 13-acetate (TPA), potent stimulators of proMMPs and TIMP-1 production, on the translation of proMMP-3 and TIMP-1 mRNAs. When human uterine cervical fibroblasts were treated with IL-1 alpha or TPA for 2h, their translations were not augmented, whereas the steady-state levels of proMMP-3 and TIMP-1 mRNAs in the cells treated with these stimuli for 24 h were increased 13.3- and 1.3-fold by IL-1 alpha and 52.5- and 5.7-fold by TPA, respectively. By contrast, transforming growth factor alpha(TGF alpha), which also binds to EGF-receptor, enhanced their production as early as 2 h after treatment, indicating that growth factors that bind to EGF-receptor are likely to be involved in the translational enhancement of proMMP-3 and TIMP-1 mRNAs. EGF partially translocated cytoplasmic protein kinase C (PKC) to plasma membrane, but the PKC down-regulation induced by 100nM TPA did not diminish the EGF-mediated translational augmentation of proMMP-3 and TIMP-1 mRNAs. In contrast, the PKC inhibitor of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) effectively suppressed the translational regulation of proMMP-3 and TIMP-1 in a dose-dependent manner during the first 2-h treatment with EGF. These results suggest that EGF and TGF alpha, but not IL-1 alpha and TPA, specifically augment the translation of proMMP-3 and TIMP-1 mRNAs and accelerate their accumulation without modifying their transcripts during the first 1-2 h treatment of human uterine cervical fibroblasts. This translational augmentation is suggested to be mediated by a TPA-insensitive atypical PKC subclass in the PKC family.

Cervix Uteri↗

RNA aptamers to mammalian initiation factor 4G inhibit cap-dependent translation by blocking the formation of initiation factor complexes.

Eukaryotic translation initiation factor 4G (eIF4G) plays a crucial multimodulatory role in mRNA translation and decay by interacting with other translation factors and mRNA-associated proteins. In this study, we isolated eight different RNA aptamers with high affinity to mammalian eIF4G by in vitro RNA selection amplification. Of these, three aptamers (apt3, apt4, and apt5) inhibited the cap-dependent translation of two independent mRNAs in a rabbit reticulocyte lysate system. The cap-independent translation directed by an HCV internal ribosome entry site was not affected. Addition of exogenous eIF4G reversed the aptamer-mediated inhibition of translation. Even though apt3 and apt4 were selected independently, they differ only by two nucleotides. The use of truncated eIF4G variants in binding experiments indicated that apt4 (and probably apt3) bind to both the middle and C-terminal domains of eIF4G, while apt5 binds only to the middle domain of eIF4G. Corresponding to the difference in the binding sites in eIF4G, apt4, but not apt5, hindered eIF4G from binding to eIF4A and eIF3, in a purified protein solution system as well as in a crude lysate system. Therefore, the inhibition of translation by apt4 (and apt3) is due to the inhibition of formation of initiation factor complexes involving eIF4A and eIF3. On the other hand, apt5 had a much weaker affinity to eIF4G than apt4, but inhibited translation much more efficiently by an unknown mechanism. The five additional aptamers have sequences and predicted secondary structures that are largely different from each other and from apt3 through apt5. Therefore, we speculate that these seven sets of aptamers may bind to different regions in eIF4G in different fashions.

Animals↗

mRNA with a <20-nt poly(A) tail imparted by the poly(A)-limiting element is translated as efficiently in vivo as long poly(A) mRNA.

The poly(A)-limiting element (PLE) is a conserved sequence that restricts the length of the poly(A) tail to <20 nt. This study compared the translation of PLE-containing short poly(A) mRNA expressed in cells with translation in vitro of mRNAs with varying length poly(A) tails. In transfected cells, PLE-containing mRNA had a <20-nt poly(A) and accumulated to a level 20% higher than a matching control without a PLE. It was translated as well as the matching control mRNA with long poly(A) and showed equivalent binding to polysomes. Translation in a HeLa cell cytoplasmic extract was used to examine the impact of the PLE in the context of varying length poly(A) tails. Here the overall translation of +PLE mRNA was less than control mRNA with the same length poly(A), and the PLE did not overcome the effect of a short poly(A) tail. Because poly(A)-binding protein (PABP) is a dominant effector of poly(A)-dependent translation we reasoned excess PABP in our extract might overwhelm PLE regulation of translation. This was confirmed by experiments where PABP was inactivated with poly(rA) or Paip2, and the effect of both treatments was reversed by addition of recombinant PABP. These data indicate that the PLE functionally substitutes for bound PABP to stimulate translation of short poly(A) mRNA.

Animals↗

Hexokinase 2 is an RNA-binding protein that regulates mRNA translation independently of glycolysis and induces melanoma cell proliferation.

Although metabolic benefits of glycolysis have been extensively described in tumor cells, the extra-metabolic functions linked to this energetic pathway in tumor growth and cell proliferation have not been clearly established yet. Recently, some key glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase 2, were reported to regulate mRNA translation. Translational control of gene expression is considered as a critical effector in cancer biology, representing a highly promising area of research. Here, we report that Hexokinase 2 (HK2), a glucose kinase that catalyzes the first step of glycolysis at the outer mitochondrial membrane (OMM), is an RNA-binding protein (RBP) that regulates mRNA translation in melanoma cell lines. Polysome profiling experiments followed by RNA sequencing indicate that the translational regulation exerted by HK2 is partly independent of the metabolic status or the glycolytic pathway. We found that HK2 specifically regulates translation of the mRNA encoding SOX10, a transcription factor implicated in the regulation of tumor initiation, maintenance, and progression in melanoma. RNA-protein interaction assays, including CrossLinking ImmunoPrecipitation (CLIP), indicate that HK2 is an RBP whose interaction with RNA is independent of its enzymatic activity, its ability to bind glucose or its association with the OMM. HK2 directly interacts with the 5' untranslated region (5'UTR) of the SOX10 mRNA through a stem-loop RNA secondary structure. Using RNA-protein proximity ligation assays and a fluorescence-based ribosome-bound mRNA mapping method, we found that high glucose conditions, which promote the release of HK2 from the OMM, induce an increase in HK2-SOX10 mRNA interaction and SOX10 mRNA translation in the cytoplasm. We further showed that HK2-dependent SOX10 mRNA translation is involved in melanoma cell proliferation and colony formation. Collectively, our data highlight a nonmetabolic function of HK2 acting as an RBP and translation regulator.

Hexokinase↗

Widespread release of translational repression across Plasmodium's host-to-vector transmission event.

Malaria parasites must respond quickly to environmental changes, including during their transmission between mammalian and mosquito hosts. Therefore, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. While the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, changes in the spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that ~200 transcripts are released for translation soon after fertilization, including those encoding essential functions. Moreover, we identified that many transcripts remain repressed beyond this point. TurboID-based proximity proteomics of the DOZI/CITH/ALBA regulatory complex revealed substantial spatial and/or compositional changes across this transmission event, which are consistent with recent, paradigm-shifting models of translational control. Together, these data provide a model for the essential translational control mechanisms that promote Plasmodium's efficient transmission from mammalian host to mosquito vector.

Animals↗

EV-D68 cleaves LARP1 and PABPC1 by 3Cpro to redirect host mRNA translation machinery toward its genomic RNA.

Enterovirus D68 (EV-D68) is an emerging pathogen associated with severe respiratory diseases and neurological complications, such as acute flaccid myelitis. EV-D68 has developed sophisticated mechanisms to hijack host translation machinery, facilitating its replication and impairing host mRNA translation. In this study, we demonstrate that EV-D68 cleaves La-related protein 1 (LARP1) and poly(A)-binding protein cytoplasmic 1 (PABPC1) through its proteases 3Cpro and 2Apro. Our results indicate that overexpressing LARP1 and PABPC1 significantly inhibits EV-D68 replication and reduces the virus-mediated suppression of host translation. While both LARP1 and PABPC1 regulate translation, they exert antiviral effects through distinct mechanisms. We found that LARP1 interacts with the 5'UTR of EV-D68 RNA through its LAM domain, and this interaction is crucial for its antiviral function. LARP1 translation modulation is also influenced by the mTOR and CDK1 signaling pathways. Viral infection inhibits mTOR and CDK1 phosphorylation, which enhances LARP1's binding to viral RNA and inhibits viral translation. To counteract this inhibition, EV-D68 cleaves LARP1 through 3Cpro, thereby promoting efficient viral translation. We also investigated other enteroviruses, such as EV-A71 and CV-A16, which similarly target LARP1 and PABPC1, indicating a conserved mechanism across enteroviruses. Our findings offer new insights into how EV-D68 manipulates host translation and highlight the potential of targeting LARP1 and PABPC1 for antiviral interventions.

Humans↗

Human transcortin synthesis by a cell-free translation of hepatic mRNA.

To evaluate the site of synthesis and to characterize the translated transcortin, poly (A)-containing RNA (mRNA) from human liver was translated in a cell-free system derived from rabbit reticulocyte lysate. The in vitro synthesized product was identified as transcortin by immuno-precipitation with its specific antiserum. This translated transcortin could be displaced from the antibody by unlabeled purified transcortin obtained from plasma. Furthermore, when the translation mixture was applied to a cortisol-Sepharose column, the translated transcortin was bound to the matrix in a specific manner, indicating that this product binds to cortisol. The molecular weight of the translated transcortin was estimated to be 45,700 by its mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis, while that of plasma transcortin was 53,800. The difference in molecular weight between the translated transcortin and plasma transcortin was probably due to the presence of pre-sequence (signal peptide) in addition to the absence of carbohydrate moiety in the former. In conclusion, human liver mRNA directed the synthesis of transcortin, and the translated transcortin binds to cortisol in spite of the absence of carbohydrate moiety.

Animals↗

A novel RNA-binding protein in neuronal RNA granules: regulatory machinery for local translation.

Local translation in neuronal dendrites is an important basis for long-term synaptic plasticity, and RNA granules in the dendrites are involved in the local translation. Here, we identify RNG105 (RNA granule protein 105), a novel RNA-binding protein, as a component of the RNA granules in dendrites of hippocampal neurons. The RNG105-localizing RNA granules contain mRNAs, the translational products of which play key roles in synaptic plasticity. RNG105 has an ability to repress translation both in vitro and in vivo, consistent with the finding that the RNA granule is translationally arrested in the basal conditions. Dissociation of RNG105 from the RNA granules is induced by BDNF, a growth factor responsible for synaptic plasticity. The RNG105 dissociation is coincident with the induction of local translation near the granules. These findings suggest that RNG105 is a translational repressor in the RNA granules and provide insight into the link between RNG105 dynamics and local translational regulation.

Amino Acid Sequence↗

A model for protein translation: polysome self-organization leads to maximum protein synthesis rates.

The genetic information in DNA is transcribed to mRNA and then translated to proteins, which form the building blocks of life. Translation, or protein synthesis, is hence a central cellular process. We have developed a gene-sequence-specific mechanistic model for the translation machinery, which accounts for all the elementary steps of the translation mechanism. We performed a sensitivity analysis to determine the effects of kinetic parameters and concentrations of the translational components on protein synthesis rate. Utilizing our mathematical framework and sensitivity analysis, we investigated the translational kinetic properties of a single mRNA species in Escherichia coli. We propose that translation rate at a given polysome size depends on the complex interplay between ribosomal occupancy of elongation phase intermediate states and ribosome distributions with respect to codon position along the length of the mRNA, and this interplay leads to polysome self-organization that drives translation rate to maximum levels.

Computer Simulation↗

Ethical hurdles for translational research.

The notion of translational research has gained considerable currency over the past few years. While such an approach promises great scientific and clinical advances, the penumbra of translational research tends to incorporate prioritizing scientific projects based upon their potential for translation; tight financial connections between sponsors, scientists and clinical investigators; and sometimes research involving biological approaches for which there is little experience determining safety. It is these aspects of translational research that raise some serious ethical challenges. In this report, we examine three specific areas that raise ethical questions: (1) the potential implications of prioritizing research objectives based on the potential for translation; (2) cautions related to moving from bench to bedside (and back again); and (3) unique questions for translational research initiatives in academic medical centers. Based on this examination, it is clear that the financial and ethical costs as well as benefits of taking a translational approach need to be considered. In the meantime, exquisite attention needs to be paid whenever translational research is likely to affect the traditional fiduciary responsibilities of scientists, clinicians and institutions to research subjects, patients and students. Successful mechanisms that might be developed to address any untoward effects should be shared and evaluated.

Clinical Trials as Topic↗

[Kinematic analysis of limitation of mouth opening of temporomandibular disorders by rotation and translation of the condyle].

PURPOSE: The purpose of this study was to determine whether rotation and translation of the condyle were useful parameters for identifying causes of limitation of mouth opening in patients with temporomandibular disorders (TMD). METHODS: In 7 healthy volunteers and 55 patients with TMD, rotation and translation of the condyle were measured using a 6 degree-of-freedom jaw movement tracking device. Patients were divided into two groups: those who could open their mouth 40 mm or greater, and those who could not open their mouth 40 mm. In each group, the relationship of rotation and translation of the condyle to maximum mouth opening, condition of the articular disc, and pain was determined. RESULTS: Both correlations between maximum mouth opening and rotation and translation of the condyle were strong, especially the former correlation. There was no marked difference in the distribution of range of mouth opening, and rotation and translation of the condyle with respect to condition of the artticular disc. Muscle pain was associated with reduced mouth opening. Even in patients without limited mouth opening, their range of mouth opening was actually reduced when they had muscle pain. Among the patients with limited mouth opening, joint pain correlated to limited translation of the condyle. However, in patients with Wo-Wo condition of articular disc, their translation of the condyle was not limited even with joint pain, but it was limited with muscle pain. CONCLUSIONS: Rotation of the condyle indicated strong correlation with muscle pain, and so is a useful parameter for estimating treatment methods by a surgical approach. On the other hand, translation of the condyle showed variations in correspondence with combinations of several factors such as opening limitation, condition of the articular disc, and pain.

Biomechanical Phenomena↗

Anterior tibial translation during different isokinetic quadriceps torque in anterior cruciate ligament deficient and nonimpaired individuals.

STUDY DESIGN: Factorial quasi-experimental design. OBJECTIVES: To quantify the effect of different levels of isokinetic concentric and eccentric knee extensor torques on the anterior tibial translation in subjects with anterior cruciate ligament (ACL) deficiency. Electromyogram (EMG) activity of 4 leg muscles was recorded in order to detect any co-activation of extensors and flexors. BACKGROUND: The rehabilitation after an ACL injury is of importance for the functional outcome of the patient. In order to construct a rehabilitation program after that injury, it is important to understand the in vivo relationships between muscle force and tibial translation. METHODS AND MEASURES: Twelve patients with unilateral ACL injury and 11 uninjured volunteers performed 36 repetitions of a quadriceps contraction at different isokinetic concentric and eccentric torque levels, on a KinCom machine (60 degrees x s(-1)), with simultaneous recordings of tibial translation (CA-4000) and EMG activity from quadriceps and hamstrings muscles. Tibial translations and EMG levels were normalized to the maximum of each subject. RESULTS: The individual anterior tibial translation increased with increased quadriceps torque in a similar manner in both quadriceps contraction modes in all legs tested. During concentric mode, translation was similar in all groups, but during eccentric mode, the mean translation was 38% larger in the ACL injured knees. No quadriceps-hamstrings co-activation occurred in any test or group. CONCLUSIONS: An ACL deficient knee can limit the translation within a normal space during concentric muscle activity but not during eccentric activity. That limitation depends on other mechanisms than hamstrings co-activation.

Adolescent↗

Translations of the humerus in persons with shoulder impingement symptoms.

STUDY DESIGN: Two-group mixed-model analysis of covariance and correlation analysis. OBJECTIVES: To determine whether differences in humeral translations exist between patients with shoulder impingement symptoms and an asymptomatic comparison group, and if so, to determine if shoulder range-of-motion (ROM) measures are associated with abnormal translations. BACKGROUND: Abnormal translations of the humeral head are believed to reduce the available subacromial space and to contribute to the development or progression of shoulder impingement symptoms. These abnormal translations have also been theorized to be related to tightness of the posterior capsule and decreased shoulder ROM. METHODS AND MEASURES: Three-dimensional humeral translations were tracked in symptomatic construction workers and an asymptomatic comparison group while elevating the arm in the scapular plane under no-load, 2.3-kg, and 4.6-kg hand-load conditions. Between-group comparisons were made across 3 phases of motion (30 degrees-60 degrees, 60 degrees-90 degrees, and 90 degrees-120 degrees) and the association between humeral translations and cross-body adduction and shoulder internal rotation ROM measures were determined by Pearson correlation analysis. RESULTS: Persons with shoulder symptoms demonstrated small but significant changes in anterior-posterior translations of the humerus. These changes for the 90 degrees-120 degrees phase of humeral elevation were moderately negatively associated with available cross-body adduction ROM. CONCLUSIONS: The identified kinematic deviations are consistent with possible reductions of the subacromial space. Further study of relationships between posterior capsule tightness, rotator cuff function, and abnormal humeral translations is warranted to better delineate underlying kinematic mechanisms that may contribute to shoulder impingement symptoms and to refine rehabilitation techniques.

Adult↗

Rat fibroblasts transfected with the human 70-kDa heat shock gene exhibit altered translation and eukaryotic initiation factor 2 alpha phosphorylation following heat shock.

Heat shock inhibits translation in a wide variety of cells. After heating, eukaryotic initiation factor 2-alpha (eIF-2 alpha) becomes phosphorylated which prevents the binding of Met-tRNA to the 40s ribosomal subunit inhibiting initiation of translation. Thermotolerant cells demonstrate resistance to inhibition of translation by additional heating suggesting that heat shock proteins may help to maintain translational integrity following thermal stress. Here we have examined the effects of increased intracellular levels of hsp70 protein on translation and eIF-2 alpha phosphorylation using rat fibroblasts stably transfected with a cloned human hsp70 gene. We observed a decrease in the rate of translational inhibition following heat shock in both hsp70-transfected and thermotolerant cells. Upon recovery at 37 degrees C, both hsp70-transfected and thermotolerant cells exhibit a faster rate of translational recovery. Utilizing slab gel isoelectric focusing coupled with immunoblotting we demonstrate that 45 degrees C heat shock leads to a rapid 4-5-fold increase in eIF-2 alpha phosphorylation, with little difference seen between control cells and hsp70-transfected cells. However, dephosphorylation of eIF-2 alpha occurs faster in the hsp70-transfected cells. These results suggest that hsp70 may play a role in facilitating the dephosphorylation of eIF-2 alpha as well as reversing the inhibition of translation following heat shock.

Animals↗

Translational vestibulo-ocular reflex evoked by a "head heave" stimulus.

The gain and symmetry of vestibulo-ocular reflexes for high-frequency, high-acceleration movements of the head are altered following unilateral vestibular lesions. These changes have been well characterized for rotational head movements (thrusts), and provide reliable markers of dysfunction in individual semicircular canals. Alterations in the vestibulo-ocular reflex (VOR) evoked by lateral, whole-body translations have also been observed. In an approach directed at the development of a bedside test of otolith function, we have recorded (scleral search coil) the VOR evoked by brief, high-acceleration lateral translations (heaves). We delivered these stimuli manually and also developed a "head sled" device that minimizes any rotational contaminating component of the stimulus. Our geometrical analysis of the stimuli enables us to take into account the translational and rotational components of the movement, and to calculate an ideal response required for stabilization of images on the fovea at different fixation distances. We observed a tracking response (visually assisted VOR) that was close to ideal for image stabilization when these methods were used to analyze responses to slow, low-amplitude lateral translations of the head. When applied to rapid, high-acceleration (0.5 g) translations, the VOR was found to be less than compensatory in subjects with normal vestibular function. In a patient with unilateral vestibular hypofunction following intratympanic gentamicin injections, both the rotational and the translational VOR were asymmetric. Responses for translations toward the treated side had lower gain than those for translations toward the normal side. These findings provide a basis for further development of this technique as a clinical test and as a method for quantitative evaluation of otolith function.

Adult↗

[Dosimetry of translating scanning beam irradiation].

BACKGROUND & OBJECTIVES: It was difficult to get a good tissue dose distribution using fixed field irradiation technique in large field. But the translating scanning beam irradiation can make it. This study was designed to create a treatment method and to evaluate the dosimetry of translating scanning beam irradiation and the clinical indications. MATERIALS & METHODS: The dosage parameter was measured under the status of the translating scanning beam irradiation in the cobalt radiotherapy machine using a translating scanning beam irradiation device which was developed by Zhejiang Cancer Hospital and Zhejiang University. The ionization chamber method was used in the dosage measurement. RESULTS: (1) The translating scanning beam irradiation can increase the percentage depth dose. (2) The dosage homogeneous distribution is better in the translating scanning beam irradiation than fixed field irradiation. (3) There is no problem in dosage connection between two closed fields in one target volume. (4) The moving penumbra was produced in the translating scanning beam irradiation. CONCLUSIONS: According to the dosage distribution property of translating scanning beam irradiation, it has some obvious advantages in big fields irradiation (for example: total spinal cord, half body, and total body irradiation).

Humans↗