SEARCH · PubMed Health

Results for “Translating”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 523 records · Page 29Linked to original sources

PubMedDate not supplied

Reversible inhibition of translation by Xenopus oocyte-specific proteins.

A characteristic of growing oocytes of all animal species is the synthesis and accumulation of messenger RNA which is destined to be used primarily by the early embryo. The mechanism(s) which regulates the translation of this maternal mRNA remains unknown. However, the inability of the oocyte to translate all of its putative mRNA has been attributed to at least three limitations: (1) The rate of translation is limited by the availability of components of the translational apparatus other than mRNA, (2) the structural organization of the mRNA prevents translation, and (3) proteins associated with the mRNA prevent translation. Several investigators have suggested that proteins associated with maternal mRNA suppress translation in sea urchin eggs, although others claim that such results may be due to experimental artefacts. Oocyte-specific proteins have been identified in association with non-translating poly(A)+ mRNAs from Xenopus laevis oocytes, and we report here that when these proteins are reconstituted with mRNAs in vitro the translation of the mRNAs in vitro is reversibly repressed. The implication is that these proteins are involved in the regulation of translation of stored maternal mRNAs.

Animals↗

PubMed2006

A nucleo-cytoplasmic SR protein functions in viral IRES-mediated translation initiation.

A significant number of viral and cellular mRNAs utilize cap-independent translation, employing mechanisms distinct from those of canonical translation initiation. Cap-independent translation requires noncanonical, cellular RNA-binding proteins; however, the roles of such proteins in ribosome recruitment and translation initiation are not fully understood. This work demonstrates that a nucleo-cytoplasmic SR protein, SRp20, functions in internal ribosome entry site (IRES)-mediated translation of a viral RNA. We found that SRp20 interacts with the cellular RNA-binding protein, PCBP2, a protein that binds to IRES sequences within the genomic RNAs of certain picornaviruses and is required for viral translation. We utilized in vitro translation in HeLa cell extracts depleted of SRp20 to demonstrate that SRp20 is required for poliovirus translation initiation. Targeting SRp20 in HeLa cells with short interfering RNAs resulted in inhibition of SRp20 protein expression and a corresponding decrease in poliovirus translation. Our data have identified a previously unknown function of an SR protein (i.e., the stimulation of IRES-mediated translation), further documenting the multifunctional nature of this important class of cellular RNA-binding proteins.

Animals↗

PubMed2006

Irreversible translation arrest in the reperfused brain.

Irreversible translation arrest occurs in reperfused neurons that will die by delayed neuronal death. It is now recognized that suppression of protein synthesis is a general response of eukaryotic cells to exogenous stressors. Indeed, stress-induced translation arrest can be viewed as a component of cell stress responses, and consists of initiation, maintenance, and termination phases that work in concert with stress-induced transcriptional mechanisms. Within this framework, we review translation arrest in reperfused neurons. This framework provides a basis to recognize that phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is the initiator of translation arrest, and a key marker indicating activation of neuronal stress responses. However, eIF2 alpha phosphorylation is reversible. Other phases of stress-induced translation arrest appear to contribute to irreversible translation arrest specifically in ischemic vulnerable neuron populations. We detail two lines of evidence supporting this view. First, ischemia, as a stress stimulus, induces irreversible co-translational protein misfolding and aggregation after 4 to 6 h of reperfusion, trapping protein synthesis machinery into functionally inactive protein aggregates. Second, ischemia and reperfusion leads to modifications of stress granules (SGs) that sequester functionally inactive 48S preinitiation complexes to maintain translation arrest. At later reperfusion durations, these mechanisms may converge such that SGs become sequestered in protein aggregates. These mechanisms result in elimination of functionally active ribosomes and preclude recovery of protein synthesis in selectively vulnerable neurons. Thus, recognizing translation arrest as a component of endogenous cellular stress response pathways will aid in making sense of the complexities of postischemic translation arrest.

Animals↗

PubMed2000

The chicken c-Jun 5' untranslated region directs translation by internal initiation.

The 5' untranslated region (UTR) of the chicken c-jun message is exceptionally GC rich and has the potential to form a complex and extremely stable secondary structure. Because stable RNA secondary structures can serve as obstacles to scanning ribosomes, their presence suggests inefficient translation or initiation through alternate mechanisms. We have examined the role of the c-jun 5' UTR with respect to its ability to influence translation both in vitro and in vivo. We find, using rabbit reticulocyte lysates, that the presence of the c-jun 5' UTR severely inhibits translation of both homologous and heterologous genes in vitro. Furthermore, translational inhibition correlates with the degree of secondary structure exhibited by the 5' UTR. Thus, in the rabbit reticulocyte lysate system, the c-jun 5' UTR likely impedes ribosome scanning resulting in inefficient translation. In contrast to our results in vitro, the c-jun 5' UTR does not inhibit translation in a variety of different cell lines suggesting that it may direct an alternate mechanism of translational initiation in vivo. To distinguish among the alternate mechanisms, we generated a series of bicistronic expression plasmids. Our results demonstrate that the downstream cistron, in the bicistronic gene, is expressed to a much higher level when directly preceded by the c-jun 5' UTR. In addition, inhibition of ribosome scanning on the bicistronic message, through insertion of a synthetic stable hairpin, inhibits translation of the first cistron but does not inhibit translation of the cistron downstream of the c-jun 5' UTR. These results are consistent with a model by which the c-jun message is translated through cap independent internal initiation. Oncogene (2000) 19, 2836 - 2845

3T3 Cells↗

PubMed2002

Inhibition of internal ribosomal entry site-directed translation of HCV by recombinant IFN-alpha correlates with a reduced La protein.

Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5'-nontranslated region (5'NTR). We investigated the effect of interferon alfa (IFN-alpha) on the IRES-directed translation of HCV, using two stably transformed cell lines, RCF-1 and RCF-26, of Huh7 cells derived from human hepatocellular carcinoma that express dicistronic reporter proteins, Renilla luciferase (RL) and firefly luciferase (FL), separated by HCV-IRES. After the administration of IFN-alpha or poly(I)-poly(C), HCV-IRES-directed translation was inhibited in a dose-dependent manner. The relative HCV-IRES activity (F/L) decreased to 60% at 5,000 IU/mL of IFN-alpha and 45% at 40 microg/mL of poly(I)-poly(C). Thus, IFN-alpha or poly(I)-poly(C) inhibited HCV-IRES-directed translation more efficiently than a cellular cap-dependent translation. 2',5'-oligoadenylate synthetase (2',5'AS) protein level in cells analyzed significantly increased after the administration of IFN-alpha, but not upon poly(I)-poly(C). Overexpression of double-stranded RNA-activated protein kinase (PKR) gene did not mimic the selective inhibition of HCV-IRES-directed translation in the transformant cells, suggesting that neither the 2',5'AS nor the PKR system are involved in this selective inhibition. Interestingly, the expression of the autoantigen, La, which has been reported to enhance HCV-IRES-directed translation, was significantly reduced after the administration of IFN-alpha and poly(I)-poly(C) in a dose-dependent manner. Transient expression of La protein completely restored the selective inhibition of HCV-IRES-directed translation by IFN-alpha and poly(I)-poly(C). These findings suggested a new antiviral mechanism induced by IFN-alpha in that IFN-alpha or poly(I)-poly(C) selectively inhibited HCV-IRES-directed translation compared with the eukaryotic cap-dependent translation through the reduction of La protein.

Apoptosis↗

PubMedDate not supplied

The effect of articular malposition after total shoulder arthroplasty on glenohumeral translations, range of motion, and subacromial impingement.

The articular surface of the normal humeral head has a variable posterior and medial offset with respect to the central axis of the humeral shaft. Recreation of the normal humeral head shaft offset is postulated to be an important consideration during shoulder arthroplasty. However, the effect of humeral head malposition is unknown. The purpose of this study was to determine the effect of articular malposition after total shoulder arthroplasty on glenohumeral translation, range of motion, and subacromial impingement. Twenty-one human cadavers were dissected and tested with the use of an active or passive shoulder model. Range of motion and translation were recorded by means of an electromagnetic tracking device. The experiment was performed in 2 phases. For kinematics study, 11 cadaver shoulders were positioned both passively and actively from maximum internal rotation to maximum external rotation at 90 degrees of total elevation in the scapular plane. Three rotator cuff and 3 deltoid muscle lines of action were simulated for active joint positioning. Passive joint positioning was accomplished with the use of a torque wrench and a nominal centering force. The testing protocol was used for the natural joint as well as for 9 prosthetic head locations: centered and 2- and 4-mm offsets in the anterior, posterior, inferior, and superior directions. Repeated-measures analysis of variance was used to test for significant differences in the range of motion and translation between active and passive positioning of the natural joint as well as all prosthetic head positions. (2) For impingement study, 10 cadaver shoulders were used in a passive model, loading the tendons of the rotator cuff with a 30-N centering force. The humerus was passively rotated from maximum internal rotation (1500 Nmm) to maximum external rotation (1500 Nmm) by means of a continuous-recording digital torque wrench. Trials were performed with the use of centered, 4-, 6-, and 8-mm offset heads in the anterior, posterior, superior, and inferior positions before and after removal of the acromion and coracoacromial ligament. The relation between change in mean peak torque (with and without acromion), passive range of motion, and humeral head offset was analyzed by means of repeated-measures analysis of variance. In the kinematics study, total range of motion and all humeral translations were greater with passive joint positioning than with active positioning (P =.01) except for total superior-inferior translation and superior-inferior translation in external rotation. Anterior to posterior humeral head offset was associated with statistically significant changes in total range of motion (P =.02), range of internal rotation (P =.02), range of external rotation (P =.0001), and total anterior-posterior translation (P =.01). Superior to inferior humeral head offset resulted in statistically significant changes in total range of motion (P =.02), range of internal rotation (P =.0001), anterior-posterior translation during external rotation (P =.01), and total superior-inferior translation (P =.03). In the impingement study, there was a significant increase in torque from centered to 4-mm inferior offset (P =.006), 6-mm inferior offset (P <.001), and 8-mm inferior offset (P <.001). There was no significant increase in torque with superior, anterior, and posterior offsets. Glenohumeral motion significantly decreased from 129 degrees for centered head to 119 degrees for 8-mm superior (P =.002), 119 degrees for 8-mm anterior (P =.014), 118 degrees for 8-mm inferior (P <.001), and 114 degrees for 8-mm posterior (P =.001). Humeral articular malposition of 4 mm or less during prosthetic arthroplasty of the glenohumeral joint may lead to small alterations in humeral translations and range of motion. Inferior malposition of greater than 4 mm can lead to increased subacromial contact; offset of 8 mm in any direction results in significant decreases in passive range of motion. Therefore if subacromial contact is to be minimized and glenohumeral motion maximized after shoulder replacement, anatomic reconstruction of the humeral head-humeral shaft offset to within 4 mm is desirable.

Adult↗

PubMed2005

Fibronectin controls cap-dependent translation through beta1 integrin and eukaryotic initiation factors 4 and 2 coordinated pathways.

Fibronectin (FN) is a major matrix protein involved in multiple processes. Little is known about how adhesion to FN affects the translational machinery. We show that in fibroblasts adhesion to FN triggers translation through the coordinated regulation of eukaryotic initiation factors (eIFs) 4F and 2 and is impaired by blocking beta1 integrin engagement. FN-stimulated translation has unique properties: (i) it is highly sensitive to the inhibition of phosphatidylinositol 3-kinase (PI3K), but not to the inhibition of mammalian target of rapamycin, downstream of PI3K; (ii) there is no synergy between serum-stimulated translation and FN-dependent translation; (iii) FN-dependent translation, unlike growth factor-stimulated translation, does not lead to increased translocation of 5' terminal oligopyrimidine tract mRNAs to polysomes; and (iv) cells devoid of attachment to matrix show an impairment of initiation of translation accompanied by phosphorylation of eIF2alpha, which cannot be reverted by active PI3K. These findings indicate that integrins may recruit the translational machinery in a unique way and that FN-dependent translation cannot be blocked by mammalian target of rapamycin inhibition.

Animals↗

PubMed2006

Expression profiling reveals meiotic male germ cell mRNAs that are translationally up- and down-regulated.

Gametes rely heavily on posttranscriptional control mechanisms to regulate their differentiation. In eggs, maternal mRNAs are stored and selectively activated during development. In the male, transcription ceases during spermiogenesis, necessitating the posttranscriptional regulation of many paternal mRNAs required for spermatozoan assembly and function. To date, most of the testicular mRNAs known to be translationally regulated are initially transcribed in postmeiotic cells. Because protein synthesis occurs on polysomes and translationally inactive mRNAs are sequestered as ribonucleoproteins (RNPs), movement of mRNAs between these fractions is indicative of translational up- and down-regulation. Here, we use microarrays to analyze mRNAs in RNPs and polysomes from testis extracts of prepuberal and adult mice to characterize the translation state of individual mRNAs as spermatogenesis proceeds. Consistent with published reports, many of the translationally delayed postmeiotic mRNAs shift from the RNPs into the polysomes, establishing the validity of this approach. In addition, we detect another 742 mouse testicular transcripts that show dramatic shifts between RNPs and polysomes. One subgroup of 35 genes containing the known, translationally delayed phosphoglycerate kinase 2 (Pgk2) is initially transcribed during meiosis and is translated in later-stage cells. Another subgroup of 82 meiotically expressed genes is translationally down-regulated late in spermatogenesis. This high-throughput approach defines the changing translation patterns of populations of genes as male germ cells differentiate and identifies groups of meiotic transcripts that are translationally up- and down-regulated.

Animals↗

PubMed2001

Internal initiation of translation of five dendritically localized neuronal mRNAs.

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5' leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the alpha subunit of calcium-calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5' untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and alpha-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5' untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5' leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.

Animals↗

PubMed1978

Inhibition of translation by poliovirus: inactivation of a specific initiation factor.

Translation of vesicular stomatitis virus (VSV) mRNA, like host mRNA translation, is inhibited in cells infected with poliovirus. To study the mechanism of poliovirus-induced inhibition of protein synthesis, we prepared extracts from poliovirus-infected and uninfected HeLa cells. Poliovirus mRNA was translated in lysates from both infected and uninfected cells, while VSV mRNA was translated only in the lysate from uninfected cells. Addition of purified translation initiation factors to the extract from infected cells showed that one factor, eIF-4B, could restore VSV mRNA translation in the infected lysate, but did not increase poliovirus mRNA translation. Further experiments involving translation of VSV mRNA in mixed extracts from poliovirus-infected and uninfected cells showed (i) that there was not an excess of an inhibitor of VSV mRNA translation in the infected lysate, but (ii) that an acitivity that caused a slow inactivation of eIF-4B was present in the infected lysate. Inactivation of eIF-4B appears to be the mechanism by which poliovirus infection causes a selective inhibition of translation.

Cell-Free System↗

PubMed1998

Repression of human thymidylate synthase mRNA translation by antisense 2'-O-methyl oligoribonucleotides.

Previous studies have shown that translation of thymidylate synthase (TS) mRNA is controlled by its own protein end product TS in a negative autoregulatory manner. Disruption of this process results in increased synthesis of TS and may be associated with the development of cellular drug resistance to TS-directed anticancer agents. As one strategy to inhibit TS expression, we have designed antisense RNA oligoribonucleotides (ORNs) that directly target the 5'-upstream binding site (nt 80-109) of TS mRNA, a critical cis-acting regulatory element. ORNs were analyzed for their ability to specifically inhibit translation of human TS mRNA in an in vitro rabbit reticulocyte lysate translation system. Native 2'-hydroxyl(OH) ORNs inhibited TS mRNA translation in a dose-dependent manner but did not repress translation of control mRNAs, including p53 or Escherichia coli TS. A control sense 2'-OH ORN was unable to repress translation of either human TS mRNA or control mRNAs. Modified antisense ORNs with 2'-O-methyl phosphodiester or 2'-O-methyl phosphorothioate backbones (or both) repressed human TS mRNA translation in a dose-dependent manner, and they were both more effective than the respective 2'-OH ORN. However, nonspecific effects on mRNA translation were observed with the 2'-O-methyl phosphorothioate ORN. In vitro translation experiments revealed that in the presence of antisense ORNs, the target TS mRNA remained intact. These findings demonstrate that antisense ORNs targeted at the 5'-upstream cis-acting element represent effective inhibitors of TS mRNA translation.

Animals↗

PubMed2003

Cell cycle-dependent translation of p27 involves a responsive element in its 5'-UTR that overlaps with a uORF.

p27(Kip1) regulates cell proliferation by binding to and modulating the activity of cyclin-dependent kinases. The CDK inhibitor is haploinsufficient for tumor suppression and reduced p27 activity is fundamental for the development of many human malignancies. Consistently, reduced p27 protein provides independent prognostic information in various tumors including breast, prostate, colon and gastric carcinomas. In normal cells, p27 protein increases in growth arrest but also oscillates during cell cycle progression. Expression of p27 is regulated through mechanisms including transcription, translation and ubiquitin-mediated degradation. Each of these pathways may contribute to deregulation of p27 in hyperproliferative diseases. p27 translation increases in proliferating cells during G(1) phase and declines as cells enter S phase. To investigate the mechanisms of p27 translational control, we analyzed fragments of the p27 transcript for their contribution to cell cycle regulated translation. We found that an element in the p27 5'-UTR can render reporter translation cell cycle sensitive with maximal translation in G1-arrested cells. This novel element of 114 nt contains a G/C-rich hairpin domain that is predicted to form multiple stable stemloops and also overlaps with a small upstream ORF (uORF). Both structures contribute to cell cycle-regulated translation. The uORF can be translated in vitro and its sequence and position are highly conserved in mice and chickens. Interestingly, the precise sequence or the length of the uORF-encoded peptide are not important for p27 translation, consistent with the idea that ribosomal recruitment to its initiation codon rather than the translation product itself contributes to the regulation.

5' Untranslated Regions↗

PubMed2001

The fragile X mental retardation protein inhibits translation via interacting with mRNA.

Fragile X syndrome is a frequent form of inherited mental retardation caused by functional loss of the fragile X mental retardation protein, FMRP. The function of FMRP is unknown, as is the mechanism by which its loss leads to cognitive deficits. Recent studies have determined that FMRP is a selective RNA-binding protein associated with polyribosomes, leading to the hypothesis that FMRP may be involved in translational regulation. Here we show that purified recombinant FMRP causes a dose-dependent translational inhibition of brain poly(A) RNA in rabbit reticulocyte lysate without accelerated mRNA degradation. In our translation reaction FMRP interacts with other messenger ribonucleoproteins and pre-exposure of FMRP to mRNA significantly increased the potency of FMRP as a translation inhibitor. Translation suppression by FMRP is reversed in a trans-acting manner by the 3'-untranslated portion of the Fmr1 message, which binds FMRP, suggesting that FMRP inhibits translation via interacting with mRNA. Consistently FMRP suppresses translation of the parathyroid hormone transcript, which binds FMRP, but not the beta-globin transcript, which does not bind FMRP. Moreover, removing the FMRP-binding site on a translation template abolishes the inhibitory effect of FMRP. Taken together, our results support the hypothesis that FMRP inhibits translation via interactions with the translation template.

Animals↗

PubMed2003

Comparison of the capacity of different viral internal ribosome entry segments to direct translation initiation in poly(A)-dependent reticulocyte lysates.

Polyadenylation stimulates translation of capped eukaryotic mRNAs and those carrying picornaviral internal ribosome entry segments (IRESes) in vivo. Rabbit reticulocyte lysates (RRL) reproduce poly(A)-mediated translation stimulation in vitro after partial depletion of ribosomes and ribosome-associated factors. Here, we have evaluated the effects of varying different parameters (extent of extract depletion, cleavage of eIF4G, concentrations of KCl, MgCl(2) and programming mRNA) on IRES-driven translation efficiency and poly(A)-dependency in ribosome-depleted RRL. For comparison, the study included a standard capped, polyadenylated mRNA. Dramatic differences were observed in the abilities of the different IRESes to direct translation in ribosome-depleted extracts. While the hepatitis A virus IRES was incapable of driving translation in physiological conditions in depleted RRL, mRNAs carrying the foot-and-mouth disease virus and hepatitis C virus IRESes were translated significantly better than a standard cellular mRNA in the same conditions. Indeed, the capacities of these IRESes to direct translation in ribosome-depleted RRL were similar to those reported previously in certain cell lines. Both the abilities of the IRESes to drive translation and their individual salt optima in ribosome-depleted extracts suggest that these elements have dramatically different affinities for some component(s) of the canonical translation machinery. Finally, using poliovirus as an example, we show that the ribosome-depleted system is well suited to the study of the translational capacity of naturally occurring IRES variants.

Animals↗

PubMed2005

The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation.

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5'-untranslated region (5'-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5'-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3'-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5'-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5'-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5'-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5'-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5'-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis.

5' Untranslated Regions↗

PubMed2006

Different modes and potencies of translational repression by sequence-specific RNA-protein interaction at the 5'-UTR.

To determine whether sequence-specific RNA-protein interaction at the 5'-untranslated region (5'-UTR) can potently repress translation in mammalian cells, a bicistronic translational repression assay was developed to permit direct assessment of RNA-protein interaction and translational repression in transiently transfected living mammalian cells. Changes in cap-dependent yellow fluorescent protein (YFP) and internal ribosome entry sequence (IRES)-dependent cyan fluorescent protein (CFP) translation were monitored by fluorescence microscopy. Selective repression of YFP or coordinate repression of both YFP and CFP translation occurred, indicating two distinct modes by which RNA-binding proteins repress translation through the 5'-UTR. Interestingly, a single-stranded RNA-binding protein from Bacillus subtilis, tryptophan RNA-binding attenuation protein (TRAP), showed potent translational repression, dependent on the level of TRAP expression and position of its cognate binding site within the bicistronic reporter transcript. As the first of its class to be examined in mammalian cells, its potency in repression of translation through the 5'-UTR may be a general feature for this class of single-stranded RNA-binding proteins. Finally, a one-hybrid screen based on translational repression through the 5'-UTR identified linkers supporting full-translational repression as well as a range of partial repression by TRAP within the context of a fusion protein.

5' Untranslated Regions↗

PubMed2000

Isolation of translationally controlled mRNAs by differential screening.

Translationalregulation plays an important role in the control of gene expression. Changes in translation initiation rates are the most common translation-regulating mechanisms, resulting in alterations in mRNA loading of ribosomes. This differential mobilization of mRNAs onto polyribosomes was used in differential screening to directly identify cDNAs whose transcripts are translationally controlled during antigenic stimulation of primary human T lymphocytes. Ribosome-free and polysome-bound mRNAs were prepared from quiescent and activated T cells and used as templates to synthesize four cDNA pools. These in turn were used as probes to hybridize four identical replicas of a T cell library or, alternatively, four cDNA arrays. Translational activation was indicated by redistribution of the hybridization signals from the ribosome-free fraction in resting T cells to the polysome-associated fraction in activated T cells. Translational repression corresponded to the opposite hybridization pattern. Fifty-two cDNAs were identified as translationally controlled by screening 472 genes in a cDNA array; 12 additional ones were obtained by screening a cDNA library. Several of the transcripts corresponded to mRNAs previously reported to be translationally controlled, thus validating the method. For the majority, however, such regulation had not yet been described. Translational control was verified for representative examples by demonstrating the redistribution of the corresponding mRNAs on polysome gradients in response to T cell activation. Our strategy therefore provides an efficient tool to directly isolate or identify translationally controlled mRNAs in a variety of physiological situations. Moreover, differential screening using arrays enables simultaneous analysis of both transcriptional and translational regulation, further enhancing the power of gene expression analysis.

Cells, Cultured↗

PubMed2006

In vivo contextual requirements for UAG translation as pyrrolysine.

Pyrrolysine and selenocysteine have infiltrated natural genetic codes via the translation of canonical stop codons. UGA translation as selenocysteine is absolutely dependent on message context. Here we describe the first experimental examination of contextual requirements for UAG translation as pyrrolysine. A hexahistidine-tagged Methanosarcina barkeri mtmB1 gene, encoding monomethylamine methyltransferase MtmB1, was introduced into Methanosarcina acetivorans. Host mtmB expression was minimized by growth on methanol and recombinant mtmB1 products monitored by anti-MtmB and anti-hexahistidine immunoblotting. UAG translation was not compromised, as recombinant MtmB1 was 1% of cellular protein with only trace UAG-terminated mtmB1 product detectable. Untranslated regions flanking mtmB1 were not required for UAG translation, but loss of a downstream pyrrolysine insertion sequence (PYLIS) significantly increased the UAG-termination product of mtmB1 and decreased the UAG-translation product, which nonetheless contained pyrrolysine. An in-frame UAG within a bacterial uidA transcript was translated in the methanogen as pyrrolysine with 20% efficiency, suggesting UAG translation in the absence of evolved context. However, predominant UAG-directed termination with enhancement of UAG translation by the PYLIS appears analogous to cis-acting elements for UGA translation as selenocysteine, although different mechanisms may underlie these recoding events.

Codon↗