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PubMedDate not supplied

Effect of leader primary structure on the translational efficiency of phosphoglycerate kinase mRNA in yeast.

In order to determine the effect of the nucleotide composition of the 5'-untranslated (leader) region on the translational efficiency of mRNA in yeast, we replaced a large part of the leader region of the phosphoglycerate kinase (PGK) gene by various deoxyoligonucleotides of defined sequence. All mutations left the context of the transcription initiation site and AUG start codon intact. The mutant genes were introduced into yeast cells on a multicopy vector and the ratio of the steady-state levels of PGK mRNA and protein were determined. We found translational efficiency to be unaffected by the presence of either an 18 nucleotides (nt) long polyA or polyC tract or by sequences consisting of mixtures of A and C residues in any proportion. In contrast, a poly U tract, as well as mixtures of U and C residues, reduced translational efficiency by a factor of two to three, presumably by long-range base-pairing between the leader and sequences elsewhere in the coding or 3'-non-coding regions of the messenger. In agreement with this hypothesis, a five-fold reduction in translational efficiency was found for an mRNA carrying a polyC tract in the leader as well as a polyG tract in the trailer, neither of which had any effect on translational efficiency by itself. Therefore, we conclude that the leader and trailer regions (including the polyA tail) of PGK mRNA are sufficiently close to base-pair when containing complementary sequences. The resulting secondary structure evidently constitutes a barrier for incoming 40S subunits on their way to the AUG start codon. The presence of an 18 nt long polyG tract in the leader completely abolished translation of the PGK mRNA in accordance with earlier observations. However, we found that leaders containing up to 40% G residues interspersed with either A or U, still allow highly efficient translation. This value is about four times as high as the average G content of leader sequences in naturally occurring yeast mRNAs. Finally, neither deletion of about 40% of the trailer sequence of PGK mRNA, nor replacement of this sequence by homopolymer tracts had any effect on translational efficiency.

Amino Acid Sequence↗

PubMed2000

Post-transcriptional regulation of rat CYP2E1 expression: role of CYP2E1 mRNA untranslated regions in control of translational efficiency and message stability.

Altered expression of hepatic CYP2E1 by xenobiotic or physiological stimuli is largely mediated through post-transcriptional mechanisms that may include altered CYP2E1 mRNA translation and/or protein degradation. Examination of the polyribosomal distribution of rat hepatic P450 mRNAs indicated that, whereas nearly all of the CYP2B, CYP3A, and CYP4A mRNAs were recovered in the polysomal fractions, indicating active translation, approximately 30-40% of CYP2E1 mRNA was not associated with polysomes and therefore not actively engaged in protein synthesis. To examine the CYP2E1 mRNA molecule for sequences that might affect its translational efficiency, a series of CYP2E1 recombinant RNAs (rcRNAs) with modified 5' or 3' untranslated regions (UTRs) was translated in vitro using the rabbit reticulocyte lysate system. Deletion of most of the CYP2E1 5' UTR, which was predicted to contain secondary structure, increased in vitro CYP2E1 protein synthesis. Polysomal distribution analyses of 5'-modified rcRNAs demonstrated that, as seen for hepatic CYP2E1 mRNA, a substantial fraction of each CYP2E1 rcRNA was not associated with polysomes. The polysomal distribution analyses of the CYP2E1 rcRNAs also confirmed that the observed changes in CYP2E1 protein synthesis were associated with altered ribosomal loading. Deletion of the poly(A) tail, and partial or complete deletion of the 3' UTR, decreased CYP2E1 protein synthesis. These changes in protein synthesis were accompanied by increased degradation of the CYP2E1 rcRNAs. Incubation with translational inhibitors, but not increased levels of RNase inhibitor, decreased the degradation of the rcRNAs during in vitro translation. In conclusion, these studies suggest that secondary structure in the 5' UTR of CYP2E1 mRNA is at least partially responsible for the inefficient translation of this mRNA. The poly(A) tail and sequences contained within the 3' UTR appear to be important for protecting CYP2E1 mRNA from RNase activity associated with the translation machinery.

3' Untranslated Regions↗

PubMed1993

Multiple controls over the efficiency of translation of the mRNAs encoding transition proteins, protamines, and the mitochondrial capsule selenoprotein in late spermatids in mice.

The mRNAs encoding protamines 1 and 2, transition proteins 1 and 2, and the mitochondrial capsule selenoprotein are translationally repressed with long poly(A) tracts in early spermatids and translationally active with heterogenous shortened poly(A) tracts in late spermatids (Kleene, 1989). In the present study, the spacing of ribosomes on the translationally active forms of each mRNA was calculated from the length of the coding region and the polysome size determined by sucrose gradient and Northern blot analysis. In addition, the rate of initiation of these five mRNAs was compared in the reticulocyte cell-free translation lysate. Our results reveal at least four additional forms of translational control over these mRNAs: (1) The vast majority of the active forms of the transition protein 1 mRNA and both protamine mRNAs sediment with polysomes in which the ribosomes are spaced closer than is typical of mammalian mRNAs (31-38 vs 80-100 bases apart). This implies that the rate of initiation is unusually fast and/or that the rate of elongation is unusually slow. (2) The mitochondrial capsule selenoprotein mRNA also initiates efficiently in vivo and in vitro, but sediments with polysomes in which the ribosomes are spaced wider than on the protamine mRNAs. The small size of these polysomes can be explained by inefficient insertion of selenocysteine residues at UGA codons. (3) The transition protein 2 mRNA is translated on small polysomes and a relatively large fraction sediments as free mRNPs in vivo. In addition, the transition protein 2 mRNA initiates translation inefficiently at high mRNA concentration and efficiently at low mRNA concentration in vitro. These observations suggest that the transition protein 2 mRNA may be translated inefficiently because it is a weak competitor for a limiting initiation factor. (4) Since low levels of cycloheximide fail to increase the polysome loading of transition protein 2 mRNA in culture, active single ribosomes may also be limited in late spermatids.

Animals↗

PubMed1998

Translational activation and cytoplasmic polyadenylation of FGF receptor-1 are independently regulated during Xenopus oocyte maturation.

FGF signaling is critical for establishing the Xenopus laevis embryonic body plan and requires the expression of functional FGF receptor during early embryogenesis. FGF receptor-1 (XFGFR) maternal mRNA is present in immature oocytes, but the protein is not expressed until oocyte maturation. In this report we demonstrate that endogenous XFGFR translation begins just prior to germinal vesicle breakdown and that translation depends on completion of earlier meiotic events. We show that the previously identified XFGFR 3'UTR translation inhibitory element (TIE), which is necessary and sufficient for repressing translation in the immature oocyte, also regulates the onset of translation during oocyte maturation. In addition we demonstrate that cytoplasmic polyadenylation of XFGFR RNA is regulated independently of TIE-mediated translation and it not sufficient to activate the translation of XFGFR. These experiments reveal that polyadenylation and translational activation are separable events in this mRNA, each of which is timed and regulated independently.

Animals↗

PubMed2002

Requirement for both EDEN and AUUUA motifs in translational arrest of Mos mRNA upon fertilization of Xenopus eggs.

Translational arrest of maternal Mos mRNA upon fertilization of Xenopus eggs is a prerequisite for the initiation of embryonic divisions. Recent studies suggest that an embryo deadenylation element (EDEN) present in the 3' untranslated region (3'UTR) is sufficient for deadenylation (and, hence, probably for translational arrest) of Mos mRNA after fertilization. By directly monitoring translation of numerous Mos mRNA constructs in Xenopus eggs, however, we show here that the EDEN is necessary but not sufficient for translational arrest of Mos mRNA. We demonstrate that two AUUUA motifs, each located solitarily and distantly from the EDEN, are also required for the translational arrest of Mos mRNA after fertilization. Significantly, translational arrest of Eg2 mRNA, another EDEN-containing maternal mRNA, also requires a single AUUUA motif located far from the EDEN. Analysis of the poly(A) tails of various Mos mRNA constructs indicates that the EDEN alone confers only partial deadenylation on Mos mRNA, and that the AUUUA motifs act to enhance EDEN-directed deadenylation in a position-dependent manner. Finally, introduction of an excess of the EDEN, but not the AUUUA motifs, into eggs can restore translation of endogenous Mos mRNA. These results suggest that the EDEN, only together with appropriately positioned AUUUA motifs and a trans-acting factor(s), can efficiently deadenylate and hence translationally arrest Mos (as well as Eg2) mRNA after fertilization.

3' Untranslated Regions↗

PubMed1997

Programmed cell death by hok/sok of plasmid R1: processing at the hok mRNA 3'-end triggers structural rearrangements that allow translation and antisense RNA binding.

The hok/sok locus of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The locus specifies two RNAs, hok mRNA and Sok antisense RNA. The post-segregational killing mediated by hok/sok is governed by a complicated control mechanism that involves both post-transcriptional inhibition of translation by Sok-RNA and activation of hok translation by mRNA 3' processing. Sok-RNA inhibits translation of a reading frame (mok) that overlaps with hok, and translation of hok is coupled to translation of mok. In the inactive full-length hok mRNA, the translational activator element at the mRNA 5'-end (tac) is sequestered by the fold-back-inhibitory element located at the mRNA 3'-end (fbi). The 5' to 3' pairing locks the RNA in an inert configuration in which the SDmok and Sok-RNA target regions are sequestered. Here we show that the 3' processing leads to major structural rearrangements in the mRNA 5'-end. The structure of the refolded RNA explains activation of translation and antisense RNA binding. The refolded RNA contains an antisense RNA target stem-loop that presents the target nucleotides in a single-stranded conformation. The stem of the target hairpin contains SDmok and AUGmok in a paired configuration. Using toeprinting analysis, we show that this pairing keeps SDmok in an accessible configuration. Furthermore, a mutational analysis shows that an internal loop in the target stem is prerequisite for efficient translation and antisense RNA binding.

Apoptosis↗

PubMed1999

Post-transcriptional control of bacteriophage T4 gene 25 expression: mRNA secondary structure that enhances translational initiation.

Secondary structure of the mRNA in the translational initiation region is an important determinant of translation efficiency. However, the secondary structures that enhance or facilitate translation initiation are rare. We have previously proposed that such structure may exist in the case of bacteriophage T4 gene 25 translational initiation region, which contains three potential Shine-Dalgarno sequences (SD1, SD2, and SD3) with a spacing of 8, 17, and 27 nucleotides from the initiation codon of this gene, respectively. We now present results that clearly demonstrate the existence of a hairpin structure that includes SD1 and SD2 sequences and brings the SD3, the most typical of these Shine-Dalgarno sequences, to a favourable spacing with the initiation codon of gene 25. Using a phage T7 expression system, we show that mutations that prevent the formation of hairpin structure or eliminate the SD3 sequence result in a decreased level of gp25 synthesis. Double mutation in base-pair V restores the level of gene 25 expression that was decreased by either of the two mutations (C-to-G and G-to-C) alone, as predicted by an effect attributable to mRNA secondary structure. We introduced the mutations into the bacteriophage T4 by plasmid-phage recombination. Changes in the plaque and burst sizes of T4 mutants, carrying single and double mutations in the translational initiation region of gene 25, strongly suggest that the predicted mRNA secondary structure controls (enhances) the level of gene 25 expression in vivo. Hybridization of total cellular RNA with a gene 25 specific probe indicated that secondary structure or mutations in the translational initiation region do not notably affect the 25 mRNA stability. Immunoblot analysis of gp25 in Escherichia coli cells infected by T4 mutants showed that mRNA secondary structure increases the level of gp25 synthesis by three- to fourfold. Since the secondary structure increases the level of gp25 synthesis and does not affect mRNA stability, we conclude that this structure enhances translation initiation. We discuss some features of two secondary structures in the translational initiation regions of T4 genes 25 and 38.

Base Sequence↗

PubMed1999

Structural and functional analysis of the 5' untranslated region of coxsackievirus B3 RNA: In vivo translational and infectivity studies of full-length mutants.

The lengthy 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) forms a highly ordered secondary structure, which plays an important role in controlling viral transcription and translation. Our previous work has delineated the internal ribosome entry site (IRES) by mutation of mono- and bicistronic plasmids containing the 5'UTR and subsequent cell- free translation in rabbit reticular lysate (D. Yang, J. E. Wilson, D. R. Anderson, L. Bohunek, C. Cordeiro, R. Kandolf, and B. M. McManus. (1997). Virology 228, 63-73). To further identify the sequence elements responsible for viral translation and infectivity in tissue culture cells, >30 full-length mutants of CVB3 were constructed by mutations of the IRES and its flanking regions. Viral RNAs were transcribed from these constructs and transfected into HeLa cells. When the stem-loops G and H in the putative IRES were deleted, viral infectivity was abolished and viral protein translation was also undetectable by immunoblot analysis. However, when stem-loops A and B were deleted or stem-loop E was partially deleted, viral protein translation could be detected although cytopathic effect could not be observed. The data suggest that the crucial sequence of the IRES is located at stem-loops G and H. Further serial deletion mapping up and down stream of the crucial sequence defined more accurately the 5' and 3' boundaries of the IRES, located at nucleotides (nts) 309-432 and 639-670, respectively. These results indicate that the core sequence of the IRES should be located at nts 432-639. This IRES segment is much shorter and located closer to the initiation codon than that of poliovirus. To further define critical nucleotides within the IRES core, site-directed mutagenesis was conducted at the IRES core sequence by PCR. A 46-nt deletion in the pyrimidine-rich tract of stem-loop G abolished viral translation and infectivity. Interestingly, five single-nt substitutions in the pyrimidine-rich tract aimed at destabilizing the base pairing between the viral IRES and host 18S rRNA did not abolish CVB3 infectivity although viral protein translation was significantly reduced. This finding suggests that ribosomal internal initiation of translation and viral infectivity not only may require RNA secondary structure but also may need tertiary structure and perhaps the assistance of host protein factors.

5' Untranslated Regions↗

PubMed2001

The 3' stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs.

Cis-acting elements that regulate translation have been identified in the 3' noncoding regions (NCRs) of cellular and viral mRNAs. As one means of analyzing the effect on translation of the conserved 3' terminal RNA structure of the West Nile virus (WNV) genome, the translation efficiencies of chimeric mRNAs composed of a CAT reporter gene flanked by viral or nonviral 5' and 3' terminal sequences were compared. In vitro, the WNV 3'(+) stem loop (SL) RNA reduced the translation efficiencies of chimeric mRNAs with either viral or nonviral 5' NCRs, suggesting that a specific 3'-5' RNA-RNA interaction was not involved. In contrast, the 3' terminal sequence of a togavirus, rubella virus, enhanced translation efficiency. The WNV 3'(+)SL reduced translation efficiency both in cis and in trans and of both capped and uncapped chimeric mRNAs. We have previously reported that three cellular proteins bind specifically to the WNV 3'(+)SL RNA. Competition between the WNV 3'(+)SL and the 5' terminus of the chimeric mRNAs for proteins involved in translation initiation could explain the translation inhibition observed.

Animals↗

PubMed2002

Hepatitis C virus IRES-dependent translation is insensitive to an eIF2alpha-independent mechanism of inhibition by interferon in hepatocyte cell lines.

Interferon (IFN) in combination with ribavirin is the main treatment for hepatitis C virus (HCV) infection. The sensitivity or resistance of the virus to IFN has been linked to certain types of the interferon sensitivity determining region (ISDR) and PKR-eIF2alpha phosphorylation homology domain (PePHD) sequences in the NS5A and E2 regions of the viral genome, respectively. In search of the other potential mechanisms of HCV resistance to IFN, we tested the effect of IFN-alpha on translational activity of the HCV IRES in various cell types. Using bicistronic dual luciferase reporter RNAs in direct RNA transfection studies, we found that the cap-dependent translation was dramatically inhibited by IFN (5- to 16-fold), whereas HCV IRES translation was inhibited only marginally in two hepatoma cell lines, Huh7 and HepG2 cells. No difference in IFN sensitivity was observed among IRESs of genotypes 1a, 1b, and 2a. Translation under the control of encephalomyocarditis virus (EMCV) IRES was inhibited by IFN to the same extent as cap-dependent translation. In cells of nonhepatic origin (HeLa and Raji), however, HCV IRES-, EMCV IRES-, and cap-dependent translation were dramatically inhibited to similar levels. The PKR expression level was enhanced by IFN in all cells, but eIF2alpha phosphorylation level was not changed, probably due to the absence of double-stranded RNA species. There was also no evidence of RNase L activation. Therefore, inhibition of translation by IFN under these conditions was probably mediated by novel IFN-induced inhibitory pathways, independent of eIF2alpha phosphorylation, while HCV IRES was not subject to this inhibition in hepatoma cell lines. Thus, HCV IRES-driven translation was resistant to IFN-induced, eIF2alpha-independent inhibition in human hepatoma cells that are frequently used in studies on HCV replication. This may present a new potential mechanism of viral resistance to IFN treatment during the early steps of virus infection.

5' Untranslated Regions↗

PubMed1994

Vir-115 gene product is required to stabilize D1 translation intermediates in chloroplasts.

The nuclear gene mutant of barley, vir-115, shows a developmentally induced loss of D1 synthesis that results in inactivation of Photosystem II. Translation in plastids isolated from 1 h illuminated vir-115 seedlings is similar to wild type. In wild-type barley, illumination of plants for 16 to 72 h results in increased radiolabel incorporation into the D1 translation intermediates of 15-24 kDa. In contrast, these D1 translation intermediates were not observed in vir-115 plastids isolated from plants illuminated for 16-72 h. In addition, after 72 h of illumination, radiolabel incorporation into D1 was undetectable in vir-115 plastids. The level and distribution of psbA mRNA in membrane-associated polysomes was similar in wild-type and vir-115 mutant plastids isolated from plants illuminated for 16-72 h. Toeprint analysis showed similar levels of translation initiation complexes on psbA mRNA in vir-115 and wild-type plastids. These results indicate that translation initiation and elongation of D1 is not significantly altered in the mutant plastids. Ribosome pausing on psbA mRNA was observed in wild-type and vir-115 mutant plastids. Therefore, the absence of D1 translation intermediates in mutant plastids is not due to a lack of ribosome pausing on psbA mRNA. Based on these results, it is proposed that vir-115 lacks or contains a modified nuclear-encoded gene product which normally stabilizes the D1 translation intermediates. In wild-type plastids, ribosome pausing and stabilization of D1 translation intermediates is proposed to facilitate assembly of cofactors such as chlorophyll with D1 allowing continued D1 synthesis and accumulation in mature chloroplasts.

Bacterial Proteins↗

PubMed1984

Translation of vaccinia virus and cellular mRNA in cell-free systems prepared from uninfected and vaccinia virus infected L929 cells.

Cell-free translation systems were prepared from uninfected and vaccinia infected (3 and 5 hours post-infection) L929 cells. The systems were made mRNA dependent in order to translate exogenous mRNA mixtures. The overall rate of protein synthesis was similar in the three translation systems. However, one-dimensional electrophoresis showed that the systems differed in terms of the translation efficiency for individual mRNAs. This could be demonstrated with each of the following mRNA mixtures: early vaccinia mRNA synthesized by vaccinia cores in vitro, mRNA isolated from polysomes of vaccinia infected HeLa cells ("late" vaccinia mRNA) and cytoplasmic ascites mRNA. When the above mentioned groups of mRNAs were allowed to compete for translation in the cell-free systems and their products were analyzed on one-dimensional gels, the following order of translational efficiency was observed: the most prominent species of vaccinia early mRNA (other species could not be judged) were translated better than some late vaccinia mRNA species which in turn were slightly more efficiently translated than cellular mRNAs.

Animals↗

PubMed1997

Deletion analysis of a translational enhancer upstream from the coat protein open reading frame of potato virus S.

We have previously demonstrated that the 101 nucleotides upstream from the ATG of the potato virus S (PVS) coat protein gene has the ability to act as a translational enhancer in vitro and in vivo when fused to a range of marker genes. These 101 nucleotides, which have been designated VTE (viral translational enhancer), contain a block of nucleotide homology that is conserved between members of the carlavirus group, centered around a core sequence of CCTTTAGGTT. When deletions were generated that lacked 47 of the 5' nucleotides, but still retained the conserved block, and analysed in vitro and in vivo, it was observed that these leaders had lost their ability to act as translational enhancers. These results suggest that the sequences 5' to the conserved block may be acting as a translational enhancer or may be important in placing the conserved block in optimal context. This is confirmed to some extent by hybrid arrest translation results in which the effects on translation of oligonucleotides, complementary to regions within the VTE leader, were investigated. It was observed that oligonucleotides complementary to the nucleotides 5' to the conserved block had a dramatic effect on the translational competence of transcripts derived from VTE-luciferase constructs, decreasing levels by 53%, whereas oligonucleotides complementary to sequences 3' to the conserved block reduced levels of translation by only 15.6%.

Base Sequence↗

PubMed2006

Evidence that PTB does not stimulate HCV IRES-driven translation.

It is now well established that Hepatitis C Virus (HCV) translation is driven by an Internal Ribosome Entry Site (IRES) resulting in cap-independent translation. Such a mechanism usually occurs with the help of IRES Associated Factors (ITAFs). Moreover, an important translational feature is likely conserved from the model of classical mRNA circularisation (5'-3' cross-talk), involving the HCV RNA highly structured 3' extremity called the 3'X region. This could bind several cellular factors and modulate the translation efficacy, at least in Rabbit Reticulocyte Lysate (RRL). In particular, polypyrimidine-binding proteins have been proposed to be potential HCV ITAFs, such as Polypyrimidine Tract Binding protein (PTB). However, contradictions still exist as to the role of PTB: its ability to bind both the HCV IRES and the 3'X region leads to the hypothesis that it could positively modulate IRES-driven translation in the presence of the X structure. Results of translational and PTB-binding studies of X mutant sequences led us to discredit PTB as protagonist of 3'X region stimulation on HCV IRES-driven translation. Moreover, competition assays of X RNA in trans on IRES-driven translation demonstrate the involvement of at least two stimulating factors and led to the conclusion that this mechanism is more complex than initially thought. Although we did not identify these factors, it is no longer doubtful that there is effectively a stimulating functional interaction between the HCV IRES and the 3'X region in RRL.

3' Untranslated Regions↗

PubMed1990

In vitro secondary structure analysis of mRNA from lacZ translation initiation mutants.

mRNA secondary structure can be an important determinant of the efficiency of translation initiation. To study the effect of secondary structure on translation initiation, in vitro secondary structure analysis was performed on 32 lacZ RNA transcripts that differ in their in vivo translation initiation efficiencies because of mutations. We have shown that well-translated RNA has a relatively unstructured translation initiation region in vitro. In contrast, the translation initiation region of many of the poorly translated RNA transcripts is involved in a stem-loop structure. Mutations that decrease the in vitro stability of the stem-loop increase the frequency of translation initiation. The sequences responsible for forming this stem-loop structure were localized to a small region of RNA. The results confirm some of the previous predictions of the RNA secondary structure of the mutant RNAs based on computer modeling, but they disagree with some of the predicted long-range interactions.

Base Composition↗

PubMed1992

Translation inhibition by an mRNA coding region secondary structure is determined by its proximity to the AUG initiation codon.

In the present study we investigate the impact of highly stable coding region secondary structures on mRNA translation efficiency. By introducing antisense segments into the 3'non-translated region of human alpha-globin mRNA we are able to synthesize a series of transcripts in which site-specific secondary structures are introduced without altering the primary structure of the 5' non-translated region, the coding region, or the encoded protein product. Coding region duplexes in close proximity to the AUG initiation codon are found to inhibit translation severely to a degree equal to that of a duplex that extends into the 5' non-translated region. In contrast, mRNAs containing duplexes positioned further 3' in the coding region translate at levels that are significantly higher although are still below those of native alpha-globin mRNA. The primary determinant of translation inhibition by coding region duplexes appears to be the proximity of the duplex to the AUG initiation codon and reflects a parallel inhibition of monosome formation. These data demonstrate that extensive coding region secondary structures suppress translation to a minimal or to a substantial degree depending on their distance from the initiation codon.

Base Sequence↗

PubMed1992

Role of cytoplasmic mRNP proteins in translation.

Polyribosomal and free mRNPs from rabbit reticulocytes were isolated and characterized. Translation of mRNPs was studied in the rabbit reticulocyte and wheat germ cell-free systems. Both classes of mRNPs were active in rabbit reticulocyte lysates. However, considerable differences between mRNPs and mRNA have been revealed. High concentrations of mRNA in the form of mRNP did not inhibit protein biosynthesis, whereas the same amounts of deproteinized mRNA caused inhibition of this process. Polyribosomal mRNPs and deproteinized mRNA, but not free mRNPs, are active in the wheat germ cell-free translation system. Translation of free mRNPs in this system can be restored by addition of 0.5 M KCl-wash of rabbit reticulocyte ribosomes. These results suggest the existence of a special repressor/activator regulatory system which controls mRNA distribution between free mRNPs and polyribosomes in rabbit reticulocytes. This regulatory system should include: i) a translation repressor associated with mRNA within free mRNPs, preventing its translation; and ii) a translation activator associated with ribosomes, overcoming the effect of the repressor. Both classes of cytoplasmic mRNPs contain a major 50 kDa protein (p50). The content of this protein per mol of mRNA in free mRNPs is twice as much as in polyribosomal ones. The method of p50 isolation has been developed and some properties of this protein were investigated. It has been shown that small amounts of p50 stimulate, whereas high amounts inhibit mRNA translation. We suggest that p50 has a dual role in protein biosynthesis. In polyribosomal mRNPs (p50:mRNA approximately 2:1, mol/mol), this protein promotes the translation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Identification of a translation inhibitory element (TIE) in the 3' untranslated region of the human interferon-beta mRNA.

We have previously reported that the 3' untranslated region (UTR) of the human interferon-beta mRNA has an inhibitory effect on the mRNA translation both in vitro, in a rabbit reticulocyte lysate, and in vivo, in the Xenopus oocyte. In the present study, we identify the sequence in the 3' UTR which is responsible for this translation inhibition. We show that this sequence is located between the 100th and 161st nucleotides downstream from the translation stop codon. It contains several repeats of the A + U-rich consensus octanucleotide UUAUUUAU, which is also present in the 3' UTR of several mRNAs involved in the inflammatory response. We also demonstrate here that the inhibitory effect of the sequence on the mRNA translation does not depend on its position in relation to the termination codon. However, no inhibition of translation is observed when this sequence is inserted in the 5' UTR of the mRNA. The removal of the translation inhibitory sequence not only improves the mRNA translation in Xenopus oocytes but it also strongly decreases the IFN-beta mRNA stability in those cells. This suggests that, in this system at least, the mRNA degradation is linked to its translational efficiency.

Animals↗