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At least 109 records · Page 6Linked to original sources

PubMed1987

3' non-translated region of human alpha-globin messenger RNA does not affect translation efficiency.

The relative translation efficiency of three synthetic alpha-globin mRNAs differing by their 3' non-translated end was measured in vitro in a rabbit reticulocyte lysate. Results showed that substituting the 3' non-translated end of human alpha 2 globin mRNA by the 3' non-translated end of chimpanzee alpha 1 or alpha 2 mRNAs has no effect on translation efficiency. In contrast, the introduction of the alpha-Quong-Sze mutation (alpha 125, Leu----Pro) in human alpha 2 mRNA led to a 50% apparent reduction in globin synthesis due to the instability of the alpha-Quong-Sze globin chain. We conclude that human alpha 1 and alpha 2 globin mRNAs have the same translation efficiency, and that the reduction, previously reported, in the kinetics of alpha-globin synthesis by alpha 2 mRNA carrying the alpha-Quong-Sze mutation is due to the instability of the alpha-Quong-Sze globin chain only.

DNA, Recombinant↗

PubMed2006

Structures required for poly(A) tail-independent translation overlap with, but are distinct from, cap-independent translation and RNA replication signals at the 3' end of Tobacco necrosis virus RNA.

Tobacco necrosis necrovirus (TNV) RNA lacks both a 5' cap and a poly(A) tail but is translated efficiently, owing in part to a Barley yellow dwarf virus (BYDV)-like cap-independent translation element (BTE) in its 3' untranslated region (UTR). Here, we identify sequence downstream of the BTE that is necessary for poly(A) tail-independent translation in vivo by using RNA encoding a luciferase reporter gene flanked by viral UTRs. Deletions and point mutations caused loss of translation that was restored by adding a poly(A) tail, and not by adding a 5' cap. The two 3'-proximal stem-loops in the viral genome contribute to poly(A) tail-independent translation, as well as RNA replication. For all necroviruses, we predict a conserved 3' UTR secondary structure that includes the BTE at one end of a long helical axis and the stem-loops required for poly(A) tail-independent translation and RNA replication at the other end. This work shows that a viral genome can harbor distinct cap- and poly(A) tail-mimic sequences in the 3' UTR.

3' Untranslated Regions↗

PubMed2003

Translational control and metastatic progression: enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs.

To form metastases, tumors must break from the primary tumor site, invade surrounding tissues, enter and survive within the circulation and ultimately colonize a distal tissue. Each of these steps requires the cooperative function of numerous proteins--proteins that facilitate angiogenesis (e.g., VEGF), cell survival (e.g., Bcl-2), invasion (e.g., MMPs), and autocrine growth stimulation (e.g., c-myc, cyclin D1). Although expression of these proteins is regulated at many levels by disparate stimuli, translation of these key malignancy-related proteins is regulated primarily by the activity of the mRNA cap-binding protein eIF-4E, the rate-limiting member of the eIF-4F translation initiation complex. By binding the cap structure at the 5' terminus of cellular mRNAs, eIF-4E recruits mRNAs to the eIF-4F complex, which then scans from the 5' cap through the untranslated region (5'UTR), unwinding secondary structure to reveal the translation initiation codon and to enable ribosome loading. Messenger RNAs with short unstructured 5' UTRs are more easily translated than mRNAs harboring lengthy, highly structured 5' UTRs, as these prohibit efficient scanning and start codon recognition. As such, the translation of these mRNAs, which typically encode proteins involved in angiogenesis (e.g., VEGF), tumor growth (cyclin D1) and survival (Bcl-2), is suppressed except when eIF-4E is engaged with the eIF-4F complex--a common event in many human and experimental cancers. This review focuses on the hypothesis that enhanced eIF-4E function contributes to metastatic progression by selectively upregulating the translation of key malignancy-related proteins that together conspire to drive the metastatic process.

Animals↗

PubMed1989

Translation of glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA is increased in poliovirus-infected cells at a time when cap-dependent translation of cellular mRNAs is inhibited.

All cellular cytoplasmic mRNAs carry a 7-methylguanylate cap attached to their 5' ends. This cap structure is recognized by cap-binding proteins that then direct the binding of ribosomal subunits to this 5'-end complex. Poliovirus, a plus-stranded RNA virus, interferes with this cellular translation process by proteolytically inactivating the cap-binding protein complex. Subsequently the viral mRNA can be translated by an initiation process in which ribosomes bind internally to the mRNA [Pelletier, J. & Sonenberg, N. (1988) Nature (London) 334, 320-325], obviating cap-dependent translation. At least one cellular mRNA, encoding a heat shock-like protein, glucose-regulated protein 78/immunoglobulin heavy-chain binding protein, has been discovered to be translated at an increased rate in poliovirus-infected cells at a time when the translation of other cellular mRNAs is inhibited. The glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA thus exemplifies a cellular mRNA that is translated at a specifically enhanced rate by an as-yet-unresolved cap-independent initiation process in cells when the cap-binding protein complex is not functional.

Carrier Proteins↗

PubMed1997

Co-translational degradation of apolipoprotein B100 by the proteasome is prevented by microsomal triglyceride transfer protein. Synchronized translation studies on HepG2 cells treated with an inhibitor of microsomal triglyceride transfer protein.

We studied the effect of inhibition of microsomal triglyceride transfer protein (MTP) on apolipoprotein (apo) B100 translation and secretion using HepG2 cells. The MTP-mediated lipid transfer activity was reduced using a specific MTP inhibitor. ApoB100 translation was synchronized by treatment with puromycin prior to L-[35S]methionine pulse-chase labeling. During the first 4 min of chase, synthesis of apoB polypeptides the size of 100-200 kDa was insensitive to the inhibitor, suggesting that inhibition of MTP did not affect the initiation of apoB100 translation. After 15 min of chase, the 100-200-kDa species were chased into polypeptides larger than 320 kDa (i.e. apoB65 or 65% of full-length apoB100) in both control and inhibitor-treated cells. However, the amount of these polypeptides decreased (by 36% for apoB65-75, by 64% for apoB75-85, by 76% for apoB85-95, and by 77% for apoB100) upon MTP inhibition. No accumulation of smaller polypeptides was observed, but total immunoprecipitable apoB radioactivity was decreased suggesting that apoB could undergo co-translational degradation when MTP activity was reduced. Inhibitors of the multicatalytic proteinase complex (proteasome) such as lactacystin or MG-115 could prevent apoB co-translational degradation. Nevertheless, MG-115 could not avoid the MTP inhibitor decreasing apoB100 secretion but rather induced the accumulation of secretion-incompetent apoB100 in the cell. These results indicate that MTP activity is required during the elongation of apoB100 polypeptides, particularly at the sequences downstream of carboxyl terminus of apoB65. Co-translational degradation might constitute a more general mechanism of early quality control for large or complex proteins.

Apolipoprotein B-100↗

PubMed2001

The yeast hsp70 homologue Ssa is required for translation and interacts with Sis1 and Pab1 on translating ribosomes.

The 70-kDa heat shock proteins are molecular chaperones that participate in a variety of cellular functions. This chaperone function is stimulated by interaction with hsp40 proteins. The Saccharomyces cerevisiae gene encoding the essential hsp40 homologue, SIS1, appears to function in translation initiation. Mutations in ribosomal protein L39 (rpl39) complement loss-of-function mutations in SIS1 as well as PAB1 (poly(A)-binding protein), suggesting a functional interaction between these proteins. However, while a direct interaction between Sis1 and Pab1 is not detectable, both of these proteins physically interact with the essential Ssa (and not Ssb) family of hsp70 proteins. This interaction is mediated by the variable C-terminal domain of Ssa. Subcellular fractionations demonstrate that the binding of Ssa to ribosomes is dependent upon its C terminus and that its interaction with Sis1 and Pab1 occurs preferentially on translating ribosomes. Consistent with a function in translation, depletion of Ssa protein produces a general translational defect that appears similar to loss of Sis1 and Pab1 function. This translational effect of Ssa appears mediated, at least in part, by its affect on the interaction of Pab1 with the translation initiation factor, eIF4G, which is dramatically reduced in the absence of functional Ssa protein.

Adenosine Triphosphatases↗

PubMed2001

Feedback inhibition of poly(A)-binding protein mRNA translation. A possible mechanism of translation arrest by stalled 40 S ribosomal subunits.

An adenine-rich cis element at the 5'-untranslated region (UTR) of Pabp1 mRNA is able to inhibit translation of its own mRNA. Similar inhibition of translation of a reporter beta-galactosidase mRNA is observed when the adenine-rich auto regulatory sequence (ARS) is placed within the 5'-UTR of this mRNA. For this translational control the distance of the ARS from the 5' cap is not important. However, it determines the number of 40 S ribosomal subunits bound to the translationally arrested mRNA. Inhibition of mRNA translation by this regulatory sequence occurs at the step of joining of the 60 S ribosomal subunit to the pre-initiation complex. Translational arrest of the ARS containing mRNA in a rabbit reticulocyte lysate cell-free system in the presence of exogenous Pabp1 protects the 5'-flanking region of the ARS from nuclease digestion. This protection depends on the binding of the 40 S ribosomal subunit to the mRNA. The size and the sequence of the nucleotide-protected fragment depends on the location of the ARS within the 5'-UTR. When the ARS is located at a distance of about 78 nucleotides from the 5' cap, a 40-nucleotide long region adjacent to the ARS is protected. On the other hand, when the ARS is moved further away from the 5' cap to a distance of approximately 267 nucleotides, a 100-nucleotide-long region adjacent to the ARS is protected from nuclease digestion. Nuclease protection is attributed to the presence of one or more stalled 40 S ribosomal subunits near the Pabp1-bound ARS.

5' Untranslated Regions↗

PubMed2002

Distinct regulation of internal ribosome entry site-mediated translation following cellular stress is mediated by apoptotic fragments of eIF4G translation initiation factor family members eIF4GI and p97/DAP5/NAT1.

Many cellular stresses lead to the inhibition of protein synthesis. Despite this, some cellular mRNAs are selectively translated under these conditions. It was suggested that the presence of internal ribosome entry site (IRES) sequences in the 5'-untranslated regions allow these mRNAs to be actively translated despite the overall cessation of protein synthesis. Here we tested the hypothesis that the IRES elements of genes that are involved in the control of cell survival are distinctly regulated by cellular stresses. We show that the transient conditions of cellular stress favor the translation of pro-survival IRES, while the severe apoptotic conditions support translation of pro-death IRES elements. Furthermore, activation of pro-death IRES during the etoposide-induced apoptosis is caspase-dependent and correlates with the expression of apoptotic fragments of two members of the eIF4G translation initiation factor family, p97/DAP5/NAT1 and eIF4GI. Our results suggest that the regulation of IRES translation during stress contributes to the fine-tuning of cell fate.

Animals↗

PubMed2005

RNA CUG-binding protein 1 increases translation of 20-kDa isoform of CCAAT/enhancer-binding protein beta by interacting with the alpha and beta subunits of eukaryotic initiation translation factor 2.

Expression of a dominant negative 20-kDa isoform of CCAAT/enhancer-binding protein (C/EBPbeta), LIP, is increased in proliferating livers and in tumor cells. Two RNA-binding proteins, CUGBP1 and calreticulin, have been implicated in the translational regulation of C/EBPbeta. In this paper, we present evidence showing several critical steps by which liver increases translation of LIP after partial hepatectomy. At early stages after partial hepatectomy, liver activates CUGBP1 by a hyperphosphorylation. The activated CUGBP1 binds to the 5' region of C/EBPbeta mRNA and replaces calreticulin, which partially represses translation of C/EBPbeta in quiescent livers. The hyperphosphorylated CUGBP1 also interacts with the alpha and beta subunits of initiation factor eIF2. Our data demonstrate that the interaction of CUGBP1 with the eIF2alpha enhances the association of CUGBP1 with ribosomes and correlates with increased translation of LIP in the liver after partial hepatectomy. Our data support the hypothesis that CUGBP1 increases translation of LIP by the interaction with the eIF2alpha subunit. This facilitates subsequent recruitment of larger numbers of ribosomes to initiate translation of LIP.

Animals↗

PubMed2005

Inhibition of host and viral translation during vesicular stomatitis virus infection. eIF2 is responsible for the inhibition of viral but not host translation.

In cells that allow replication of vesicular stomatitis virus (VSV), there are two phases of translation inhibition: an early block of host translation and a later inhibition of viral translation. We investigated the phosphorylation of the alpha subunit of the eIF2 complex during these two phases of viral infection. In VSV-infected cells, the accumulation of phosphorylated (inactivated) eIF2alpha did not begin until well after host protein synthesis was inhibited, suggesting that it only plays a role in blocking viral translation later after infection. Consistent with this, cells expressing an unphosphorylatable eIF2alpha showed prolonged viral protein synthesis without an effect on host protein synthesis inhibition. Induction of eIF2alpha phosphorylation at early times of viral infection by treatment with thapsigargin showed that virus and host translation are similarly inhibited, demonstrating that viral and host messages are similarly sensitive to eIF2alpha phosphorylation. A recombinant virus that expresses a mutant matrix protein and is defective in the inhibition of host and virus protein synthesis showed an altered phosphorylation of eIF2alpha, demonstrating an involvement of viral protein function in inducing this antiviral response. This analysis of eIF2alpha phosphorylation, coupled with earlier findings that the eIF4F complex is modified earlier during VSV infection, supports a temporal/kinetic model of translation control, where at times soon after infection, changes in the eIF4F complex result in the inhibition of host protein synthesis; at later times, inactivation of the eIF2 complex blocks VSV protein synthesis.

Animals↗

PubMed2006

Co-translational binding of GroEL to nascent polypeptides is followed by post-translational encapsulation by GroES to mediate protein folding.

The eubacterial chaperonins GroEL and GroES are essential chaperones and primarily assist protein folding in the cell. Although the molecular mechanism of the GroEL system has been examined previously, the mechanism by which GroEL and GroES assist folding of nascent polypeptides during translation is still poorly understood. We previously demonstrated a co-translational involvement of the Escherichia coli GroEL in folding of newly synthesized polypeptides using a reconstituted cell-free translation system (Ying, B. W., Taguchi, H., Kondo, M., and Ueda, T. (2005) J. Biol. Chem. 280, 12035-12040). Employing the same system here, we further characterized the mechanism by which GroEL assists folding of translated proteins via encapsulation into the GroEL-GroES cavity. The stable co-translational association between GroEL and the newly synthesized polypeptide is dependent on the length of the nascent chain. Furthermore, GroES is capable of interacting with the GroEL-nascent peptide-ribosome complex, and experiments using a single-ring variant of GroEL clearly indicate that GroES association occurs only at the trans-ring, not the cis-ring, of GroEL. GroEL holds the nascent chain on the ribosome in a polypeptide length-dependent manner and post-translationally encapsulates the polypeptide using the GroES cap to accomplish the chaperonin-mediated folding process.

Cell-Free System↗

PubMed1989

Restoration of a translational stop-start overlap reinstates translational coupling in a mutant trpB'-trpA gene pair of the Escherichia coli tryptophan operon.

The trpB and trpA coding regions of the polycistronic trp mRNA of Escherichia coli are separated by overlapping translation stop and start codons. Efficient translation of the trpA coding region is subject to translational coupling, i.e., maximal trpA expression is dependent on prior translation of the trpB coding region. Previous studies demonstrated that the trpA Shine-Dalgarno sequence (within trpB) and/or the location of the trpB stop codon influenced trpA expression. To examine the effect of stop codon location specifically, we constructed plasmids in which different nucleotide sequences preceding the trpA start codon were retained, and only the reading frame was changed. When trpB translation proceeded in the wild type reading frame and terminated at the normal trpB stop codon, trpA polypeptide levels were elevated over the levels observed when translation stopped before or after the natural trpB stop codon. The proximity of the trpB stop codon to the trpA start codon therefore markedly influences trpA expression.

Base Sequence↗

PubMed2004

Testing the conservation of the translational machinery over evolution in diverse environments: assaying Thermus thermophilus ribosomes and initiation factors in a coupled transcription-translation system from Escherichia coli.

Ribosomes from the extreme thermophile Thermus thermophilus are capable of translation in a coupled transcription-translation system derived from Escherichia coli. At 45 degrees C, T.thermophilus ribosomes translate at approximately 25-30% of the maximal rate of E.coli ribosomes, and synthesize full-length protein. T.thermophilus and E.coli subunits can be combined to effect translation, with the spectrum of proteins produced depending upon the source of the 30S subunit. In this system, T.thermophilus ribosomes function in concert with E.coli translational factors and tRNAs, with elongation and release factors being supplied from the E.coli extract, and purified initiation factors (IFs) being added exogenously. Cloned and purified T.thermophilus IF1, IF2 and IF3 supported the synthesis of the same products in vitro as the E.coli factors, although the relative levels of some polypeptides were factor dependent. We conclude that, at least between these two phylogenetically distant species, translational factor function and subunit-subunit interactions are conserved. This functional compatibility is remarkable given the extreme and highly divergent environments to which these species have adapted.

Escherichia coli↗

PubMed1993

Effects of sequence elements in the potato virus X RNA 5' non-translated alpha beta-leader on its translation enhancing activity.

The 5' non-translated alpha beta-leader sequence of potato virus X RNA consists of two regions: the alpha sequence (41 nucleotides with no G) and the beta sequence (42 nucleotides upstream from AUG). The alpha beta-leader has been shown to enhance strongly the expression of adjacent genes in chimeric mRNAs. This phenomenon has been postulated to be due to the unpaired conformation of the 5'-terminal 30 nucleotides and/or to the presence within the alpha region of the CCACC pentanucleotide complementary to the 3'-terminal conserved structure of 18S rRNA. Different derivatives of alpha beta-leader have been constructed for use in determining the contribution of separate elements of the alpha beta sequence to translational enhancement. It was found that deletion of the alpha sequence large fragment which was supposed to be unfolded did not reduce the delta alpha beta-leader enhancement activity. Moreover, translational enhancement was greater for this derivative. Deletion of the beta sequence resulted in a considerable increase in activity of the alpha-leader showing that the beta region was dispensable for translation. Disruption or 'masking' of CCACC led to inactivation of the alpha beta-leader as a translational enhancer. Thus, we identified the CCACC pentanucleotide as the primary motif responsible for the translation enhancing ability of alpha beta-leader.

Base Sequence↗

PubMed1994

Biphasic Stimulation of Translational Activity Correlates with Induction of Translation Elongation Factor 1 Subunit [alpha] upon Wounding in Potato Tubers.

Potato (Solanum tuberosum) tubers exhibit an increase in translational activity in response to mechanical wounding. The response is biphasic, with an initial stimulation apparent within the first 2 h after wounding and a second increase occurring 12 to 24 h after wounding. Increased activity is apparent by measurement of protein synthesis both in vivo and in vitro using a cell-free extract. Accumulation of the translational elongation factor 1 subunit [alpha] (EF-1[alpha]) parallels translational activity. Changes in the steady-state level of EF-1[alpha] mRNA, and expression of a chimeric EF-1[alpha] promoter/[beta]-glucuronidase construct in transgenic potato tubers, indicate that the gene encoding EF-1[alpha] is transcribed during both periods of translational stimulation. These results indicate that stimulation of translational activity is coordinated with increased expression and accumulation of translation factors.

Journal Article↗

PubMed1996

Translation of Sindbis virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation.

Alphaviruses, particularly Sinbis virus and Semliki Forest virus, are proving to be useful vectors for the expression of heterologous genes. In infected cells, these self-replicating vectors (replicons) transcribe a subgenomic mRNA that codes for a heterologous protein instead of the structural proteins. We reported recently that translation of the reporter gene lacZ is enhanced 10-fold when the coding sequences of this gene are fused downstream of and in frame with the 5' half of the capsid gene (I. Frolov and S. Schlesinger, J. Virol. 68:8111-8117, 1994). The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin structure. We have mutated this region by making changes in the codons which do not affect the protein sequence but should destabilize the putative hairpin structure. These changes caused a decrease in the accumulation of the capsid-beta-galactosidase fusion protein. When these alterations were inserted into the capsid gene in the context of the intact Sindbis virus genome, they led to a decrease in the rate of virus formation but did not affect the final yield. We also altered the original sequence to one that has 12 contiguous G.C base pairs and should form a stable hairpin. The new sequence was essentially as effective as the original had been in enhancement of translation and in the rate of virus formation. The position of the predicted hairpin structure is important for its function; an insertion of 9 nucleotides or a deletion of 9 nucleotides decreased the level of translation. The insertion of a hairpin structure at a particular location downstream of the initiating AUG appears to be a way that alphaviruses have evolved to enhance translation of their mRNA, and, as a consequence, they produce high levels of the structural proteins which are needed for virus assembly. This high level of translation requires an intracellular environment in which host cell protein synthesis is inhibited.

Amino Acid Sequence↗

PubMed1993

Translational regulation of human beta interferon mRNA: association of the 3' AU-rich sequence with the poly(A) tail reduces translation efficiency in vitro.

The 3' AU-rich region of human beta-1 interferon (hu-IFN beta) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3' AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFN beta mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3' AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFN beta mRNA. Differences in translational efficiency of the various hu-IFN beta mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFN beta mRNA failed to form large polysomes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysomes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3' AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3' AU-rich sequence may regulate the translation of hu-IFN beta mRNA by interacting with the poly(A) tail.

Animals↗

PubMed1998

Eukaryotic translation initiation factor 4G is targeted for proteolytic cleavage by caspase 3 during inhibition of translation in apoptotic cells.

Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.

Amino Acid Chloromethyl Ketones↗