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PubMed2004

Translation modulation of acid beta-glucosidase in HepG2 cells: participation of the PKC pathway.

Acid beta-glucosidase (GCase) is the enzyme deficient in Gaucher disease, a prototypical inherited metabolic error for enzyme and gene therapy. An 80 kDa mammalian cytoplasmic translational control protein (TCP80) modulates GCase translation in vitro and ex vivo by interacting with the 5' coding region of GCase RNA. Ten predicted PKC phosphorylation sites (Ser- or Thr-) are in the TCP80 protein. Phosphorylation of TCP80 in vitro by PKC greatly enhanced its translational inhibitory function using in vitro translation assays; binding of GCase mRNA to TCP80 was unaltered. Conversely, de-phosphorylation of TCP80 reduced its translational inhibitory function. Phosphorylation-related modulation of GCase mRNA translation also was studied in HepG2 cells. GCase expression (protein and activity levels) in HepG2 cells increased (>2-fold) in cells treated with bisindolylmaleimide (BIM), a highly selective PKC specific inhibitor. This correlated with a 90% reduction in TCP80 phosphorylation in the presence of BIM. The amount of TCP80 protein in cytoplasm and its RNA-binding activity were unchanged. These experiments indicate that GCase mRNA translation is modulated by PKC signaling pathways that are mediated through TCP80. These findings indicate potential broader impacts of the TCP/PKC system on expression of this and other genes of therapeutic interest.

Base Sequence↗

PubMed1998

Translational inhibition of messenger RNA of the human pi class glutathione S-transferase by antisense oligodeoxyribonucleotides.

In this study, a T7 plasmid expression vector containing the cDNA of a variant human GST-pi gene, hGSTP1*C, was used to examine the translational inhibition of the GST-pi mRNA with antisense deoxyribonucleotides (AS-ONs), and to investigate the dependency of the inhibition on ribonuclease (RNAse) H, AS-ON and target mRNA sequence specificity and AS-ON back bone modification. Translational inhibition of hGSTP1*C mRNA showed significant AS-ON concentration-dependency and was both target mRNA and AS-ON sequence specific. Fully modified phosphoromonthioate AS-ONs were less inhibitory than their partial phosphoromonthioate analogs; unmodified AS-ONs were inactive. RNAse H enhanced the translational inhibition by AS-ON specific to the translation initiation region mRNA, and was associated with cleavage of the target mRNA at the site of AS-ON:mRNA hybridization. AS-ONs directed to the A-->G and C-->T transitions, unique to hGSTP1*C, were more RNAse H-dependent than AS-ONs directed against the translation initiation site, indicating a greater involvement of RNAse H-dependent mRNA cleavage in the mechanism of translational inhibition by AS-ON at the polymorphic site. These data suggest that AS-ONs provide a potentially effective means of specific down-regulation of the human GST-pi gene, and demonstrate that the sites of GST-pi gene allelo-polymorphism can be targeted to translationally down-regulate the different GST-pi gene variants, specifically and differentially targeted.

Base Sequence↗

PubMed1997

Translational control of terminal oligopyrimidine mRNAs requires a specific regulator.

Terminal oligopyrimidine (TOP) mRNAs are a group of messengers translationally regulated according to the growth status of the cell. Two hypotheses have been proposed for the mechanism of the regulation: (i) there is a specific translational regulator which can reversibly alter TOP-mRNA structure, (ii) a component of the general translational apparatus can specifically affect the translation of TOP-mRNAs. To verify one of the two hypotheses we induced a partial inhibition of translation initiation in Xenopus cultured cells and analyzed the effect on TOP-mRNA translation. Our results suggest that a specific regulator is necessary to explain the translational control of these of mRNAs.

Animals↗

PubMed2000

Cell cycle regulation of hepatitis C virus internal ribosomal entry site-directed translation.

BACKGROUND & AIMS: Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5' nontranslated segment of the viral RNA. This RNA segment is known to form binary complexes with isolated 40S ribosome subunits in vitro, but there is little understanding of how the process of virus translation is regulated in vivo. METHODS: We established 2 stably transformed cell lines from Huh-7 cells that constitutively express dicistronic RNA transcripts containing sequences encoding 2 reporter proteins (Renilla luciferase and firefly luciferase) separated by a functional HCV IRES. The translation of the upstream Renilla luciferase reading frame is initiated in these cells by the usual cellular cap-dependent mechanism, whereas translation of the downstream firefly luciferase reading frame is initiated by the IRES. RESULTS: Compared with cap-dependent translation, the activity of the IRES was greatest in actively growing cells and relatively reduced in resting cells. In synchronized cultures of these stably transformed cells, the IRES activity varied with the cell cycle and was greatest during the mitotic (M) phases and lowest during the quiescent (G(0)) phases. CONCLUSIONS: These findings suggest that HCV translation is regulated by cellular proteins that vary in abundance during the cell cycle and that viral translation may be enhanced by factors that stimulate the regeneration of hepatocytes in patients with chronic hepatitis C.

5' Untranslated Regions↗

PubMed2002

Uncoupling of initiation factor eIF5B/IF2 GTPase and translational activities by mutations that lower ribosome affinity.

Translation initiation factor eIF5B/IF2 is a GTPase that promotes ribosomal subunit joining. We show that eIF5B mutations in Switch I, an element conserved in all GTP binding domains, impair GTP hydrolysis and general translation but not eIF5B subunit joining function. Intragenic suppressors of the Switch I mutation restore general translation, but not eIF5B GTPase activity. These suppressor mutations reduce the ribosome affinity of eIF5B and increase AUG skipping/leaky scanning. The uncoupling of translation and eIF5B GTPase activity suggests a regulatory rather than mechanical function for eIF5B GTP hydrolysis in translation initiation. The translational defect suggests eIF5B stabilizes Met-tRNA(i)(Met) binding and that GTP hydrolysis by eIF5B is a checkpoint monitoring 80S ribosome assembly in the final step of translation initiation.

Alleles↗

PubMed2003

Differential translation of TOP mRNAs in rapamycin-treated human B lymphocytes.

TOP mRNAs (contain a 5' terminal oligopyrimidine tract) are differentially translated in rapamycin-treated human B lymphocytes. Following rapamycin treatment, ribosomal protein (rp) and translation elongation factor TOP mRNAs were translationally repressed, whereas hnRNP A1 TOP mRNA was not. Poly(A)-binding protein (Pabp1) TOP mRNA was translationally repressed under all conditions tested. To investigate the mechanism involved, chimeric mRNAs containing the hnRNP A1 5' untranslated region (UTR) linked to the human growth hormone (hGH) reporter were analyzed. Wild-type hnRNP A1 construct mRNA behaved similarly to endogenous hnRNP A1, whereas a single mutation (guanosine to cytidine) within the TOP element resulted in increased translational regulation. These results suggest that TOP mRNA translation can be modulated and that all TOP mRNAs are not translated with equal efficiency.

5' Untranslated Regions↗

PubMed1999

Problems translating a questionnaire in a cross-cultural setting.

Questionnaires are widely used in veterinary epidemiological studies. With careful design, assessment and administration, questionnaires can collect accurate data. In cross-cultural settings, questionnaire design is complicated by the added step of translation. As part of a cross-sectional study of smallholder pig raisers in the Philippines, we assessed the importance of translation as a source of error in our questionnaire. The questionnaire contained 120 questions that collected data for entry into 361 separate data-entry fields in a computerised database. The translation was verified using the methods of back-translation and pilot-testing to identify and quantify errors of literal translation, omission and mistranslation. Errors were identified in 44 (37%) questions that obtained data for 147 (41%) data-entry fields. Although errors might have remained in the final translated version of the questionnaire, we have no doubt that the verification procedures substantially improved the internal validity of the questionnaire. It is important that veterinary epidemiologists use such procedures to check the internal validity of translated questionnaires.

Animal Husbandry↗

PubMed1995

Extrinsic wrist ligaments in the pathomechanics of ulnar translation instability.

A description of the various combinations of ligament disruptions necessary for ulnar translation instability is presented based on a series of cadaver dissections and load studies. Dissections and load studies demonstrated that major ligament disruption, even to the extent that only the dorsal ulnolunate, the palmar ulnolunate or the radioscaphocapitate, and the long radiolunate ligaments were left intact, failed to result in meaningful ulnar translation of the carpus. The radioscaphocapitate ligament alone could not prevent significant ulnar translation. Palmar translation instability was evident with less ligament disruption than that needed for ulnar translation and was always evident if there was ulnar translation. These studies imply that ulnar translation represents a much more global ligament disruption and instability than previously suggested in the literature.

Aged↗

PubMed2002

A new translational repression element and unusual transcriptional control regulate expression of don juan during Drosophila spermatogenesis.

The Drosophila don juan (dj) gene encodes a basic protein that is expressed solely in the male germline and shows structural similarities to the linker histone H1. Don Juan is located in two different subcellular structures: in the nucleus during the phase of chromatin condensation and later in the mitochondrial derivatives starting with spermatid individualization. The don juan gene is transcribed in primary spermatocytes under the control of 23 bp upstream in combination with downstream sequences. During meiotic stages and in early spermatid stages don juan mRNA is translationally repressed for several days. Analysis of male sterile mutants which fail to undergo meiosis shows that release of dj mRNA from translational repression is independent of meiosis. In gel retardation assays 60 nucleotides at the end of the dj leader form four major complexes with proteins that were extracted from testes but not with protein extracts from ovaries. Transformation studies prove that in vivo 35 bp within that region of the dj mRNA is essential to confer translational repression. UV cross-linking studies show that a 62 kDa protein specifically binds to the same region within the 5' untranslated region. The dj translational repression element, TRE, is distinct from the translational control element, TCE, described earlier for all members of the Mst(3)CGP gene family. Moreover, expression studies in several male sterile mutants reveal that don juan mRNA is translated in earlier developmental stages during sperm morphogenesis than the Mst(3)CGP mRNAs. This proves that translational activation of dormant mRNAs in spermatogenesis occurs at different time-points which are characteristic for each gene, an essential feature for coordinated sperm morphogenesis.

5' Untranslated Regions↗

PubMed1999

Translational control of nuclear lamin B1 mRNA during oogenesis and early development of Xenopus.

Cytoplasmic polyadenylation of specific mRNAs is commonly correlated with their translational activation during development. A canonical nuclear polyadenylation element AAUAAA (NPE) and cytoplasmic polyadenylation element(s) (CPE) are necessary and sufficient for polyadenylation during egg maturation. We have characterized cis-acting sequences of Xenopus nuclear lamin B1 mRNA that mediate translational regulation. By injection of synthetic RNAs into oocytes we show that the two CPE-like elements found in the 3'-untranslated region of B1 mRNA act as translational repressors in oocytes. The same CPEs in conjunction with the NPE confer transient polyadenylation and translational activation during egg maturation. Poly(A) length determination of the endogenous lamin B1 mRNA reveals a gradual increase of poly(A) tail length in early development up to mid-blastula, and a shortening of poly(A) tails during gastrulation and neurulation. The same kinetic and extent of polyadenylation and poly(A) tail shortening is observed with synthetic RNAs injected into fertilized eggs. Polyadenylation and translational activation of these RNAs is independent of the two CPEs and a NPE during early development. While translational regulation of lamin B1 mRNA functions in parts via established mechanisms, the pattern of polyadenylation and deadenylation during early development points to a novel mode of translational regulation.

3' Untranslated Regions↗

PubMed1996

Mechanisms of translational control in early development.

Oocytes accumulate a dowry of maternal mRNAs in preparation for embryogenesis. These maternal transcripts are kept dormant until late oogenesis or early embryogenesis when their translation is activated. In recent years, three types of translational control acting on maternal mRNAs have emerged: translational activation by cytoplasmic polyadenylation, translational activation by RNA localization, and regulated translational repression. In each case, translational control depends on the binding of trans-acting factors to sequences in the 3' untranslated region (3'UTR). Identification of these trans-acting factors is beginning to shed light on the molecular mechanisms that mediate translational control.

Animals↗

PubMed2000

Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation.

The poly(A)-binding protein Pab1p interacts directly with the eukaryotic translation initiation factor 4G (eIF4G) to facilitate translation initiation of polyadenylated mRNAs in yeast [1,2]. Although the eIF4G-PABP interaction has also been demonstrated in a mammalian system [3,4], its biological significance in vertebrates is unknown. In Xenopus oocytes, cytoplasmic polyadenylation of several mRNAs coincides with their translational activation and is critical for maturation [5-7]. Because the amount of PABP is very low in oocytes [8], it has been argued that the eIF4G-PABP interaction does not play a major role in translational activation during oocyte maturation. Also, overexpression of PABP in Xenopus oocytes has only a modest stimulatory effect on translation of polyadenylated mRNA and does not alter either the efficiency or the kinetics of progesterone-induced maturation [9]. Here, we report that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces translation of polyadenylated mRNA and dramatically inhibits progesterone-induced maturation. Our results show that the eIF4G-PABP interaction is critical for translational control of maternal mRNAs during Xenopus development.

Amino Acid Sequence↗

PubMed2002

Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation.

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60-100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90-156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.

5' Untranslated Regions↗

PubMedDate not supplied

Glenohumeral joint translation after arthroscopic thermal capsuloplasty of the posterior capsule.

Treatment of recurrent posterior or multidirectional glenohumeral instability in athletes with traditional operative management has produced variable results at long-term follow-up. The purpose of this study was to determine whether an arthroscopic thermal capsuloplasty of the posterior capsule with a radiofrequency probe significantly decreases anterior-posterior glenohumeral translation. Successive posterior and anterior loads of 10, 15, and 20 N were applied sequentially to 7 cadaveric shoulder joints that were mounted in a translation testing apparatus with an electromagnetic tracking device measuring posterior and anterior glenohumeral translation. Arthroscopic thermal capsuloplasty was then performed on the posterior capsular tissue with a radiofrequency probe. The identical posterior-anterior loading protocol was then repeated, and translations were recorded. The results demonstrated no statistically significant differences in the mean posterior translation measurements before and after arthroscopic thermal capsuloplasty of the posterior capsule for the 10-N (+8.9%), 15-N (-3.1%), or 20-N (-1.8%) load (P >.50 to.62). Slightly greater changes occurred in anterior translation after posterior capsuloplasty at 10 N (-1.0%), 15 N (-6.0%), and 20 N (-10.3%). However, these changes were not found to be significant either (P =.06 to.62). The results of this study demonstrated that neither posterior nor anterior glenohumeral translation was significantly decreased by thermal capsuloplasty of the posterior capsule. Perhaps the lack of substantial collagenous material in the thin posterior capsule accounts for the inability of thermal capsuloplasty to be effective in this region.

Arthroscopy↗

PubMedDate not supplied

Translation of the glenohumeral joint in patients with anterior instability: awake examination versus examination with the patient under anesthesia.

Fifty patients with a clinical diagnosis of traumatic anterior shoulder instability underwent bilateral shoulder translation testing while both awake and under anesthesia. Each patient was examined by 2 surgeons following guidelines developed by the American Shoulder and Elbow Surgeons. A single translation grade was established for anterior, posterior, and inferior directions. A comparison of means was performed with a paired t test. The mean anterior translation grade was significantly higher on the affected side when compared with that of the unaffected side both during awake examination and during examination with the patient under anesthesia (EUA). Ipsilateral comparison revealed significantly greater translation for both affected and unaffected shoulders in anterior, posterior, and inferior directions during EUA than during awake examination. Side-to-side comparison of posterior and inferior translation was similar for both awake examination and EUA. Clinical translation testing was helpful in the diagnosis of anterior shoulder instability. Side-to-side differences were subtle while awake and more apparent during EUA. The usefulness of awake translation testing for traumatic anterior instability was not clearly demonstrated; however, EUA provides helpful information to confirm the direction and degree of instability.

Adult↗

PubMed2000

Overlapping but distinct RNA elements control repression and activation of nanos translation.

Spatially restricted synthesis of Nanos protein in the Drosophila embryo is essential for anterior-posterior patterning. Nanos translation is restricted to the posterior of the embryo by translational repression of nanos mRNA throughout the bulk cytoplasm and selective activation of posteriorly localized nanos mRNA. A 90-nucleotide translational control element (TCE) mediates translational repression. We show that TCE function requires formation of a bipartite secondary structure that is recognized by Smaug repressor and at least one additional factor. We also demonstrate that translational activation requires the interaction of localization factors with sequences that overlap TCE structural motifs. The identification of separate but overlapping recognition motifs for translational repressors and localization factors provides a molecular mechanism for the switch between translational repression and activation.

3' Untranslated Regions↗

PubMed1999

Inhibition of 6-methyladenine formation decreases the translation efficiency of dihydrofolate reductase transcripts.

Cycloleucine was used to inhibit the formation of internal N6-methyladenosine residues in the messenger ribonucleic acid transcripts from cultured methotrexate resistant mouse sarcoma cells. Cells cultured in cycloleucine produced transcripts deficient in N6-methyladenosine residues and the 2'-O-methylated nucleosides of the cap structure; however, the formation of the 7-methylguanine nucleoside of the cap was not effected. Cytoplasmic polyadenylated transcripts were isolated from cells which had been pretreated with media containing cycloleucine and translated in an in vitro translation assay. The levels of translated dihydrofolate reductase were then analyzed by polyacrylamide gel electrophoresis. The amount of dihydrofolate reductase protein produced from the transcripts of the cycloleucine treated cells was 20% less than untreated transcripts. Ribonuclease protection assays demonstrated little difference in the cytoplasmic levels of dihydrofolate reductase transcripts between treated and untreated cells suggesting that the decrease in translation efficiency was not caused solely by an alteration in the processing or cytoplasmic transport of the transcripts. Translation of in vitro transcribed transcripts showed the presence of 2'-O-methylated nucleosides in the cap structure had a negative effect on translation efficiency, demonstrating that the results observed from cycloleucine treatment could not be due to the inhibition of 2'-O-methylation in the cap. These experiments therefore suggest that an inhibition of N6-methyladenosine residues in dihydrofolate reductase transcripts significantly alters their rate of translation.

Adenine↗

PubMed2000

Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation.

In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.

5' Untranslated Regions↗