SEARCH · PubMed Health

Results for “translational efficiency”

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 91 records · Page 5Linked to original sources

PubMed1996

Internal 6-methyladenine residues increase the in vitro translation efficiency of dihydrofolate reductase messenger RNA.

N6-Methyladenosine (m6A) is found internally in a number of mRNA molecules from higher eucaryotic cells. In these investigations, it was found that the presence of m6A residues increase the in vitro translation efficiency of capped T7 transcripts of mouse dihydrofolate reductase (DHFR) mRNA. Using an in vitro rabbit reticulocyte translation system, the formation of internal m6A residues in the DHFR transcripts resulted in a 1.5-fold increase in translated DHFR compared to transcripts void of internal m6A residues. Translation in a wheat germ system, however, resulted in no increase in translation efficiency upon m6A formation, suggesting that the mechanism may be species-specific.

Adenine↗

PubMed2005

The leader intron of Arabidopsis thaliana genes encoding cytochrome c oxidase subunit 5c promotes high-level expression by increasing transcript abundance and translation efficiency.

The involvement of regions located upstream of the translation start site in the expression of two Arabidopsis thaliana nuclear COX5c genes encoding subunit 5c of mitochondrial cytochrome c oxidase has been analysed. It was observed that these regions, which include a leader intron, direct the tissue-specific expression of the gus reporter gene, mainly in root and shoot meristems, actively growing tissues and vascular strands. Expression was also observed in flowers, specifically localized in anthers, stigma, and the receptacle, and in developing seeds. GUS activity measurements in protein extracts from transformed plants indicated that expression levels are higher than those observed with the constitutive CaMV 35S promoter. Removal of the leader intron produced a significant decrease in expression to values only slightly higher than those observed with a promoterless gus gene. Histochemical staining of plants transformed with the intronless construct revealed expression only in pollen, suggesting that regulatory elements capable of directing pollen-specific expression are present upstream of the intron. The COX5c-2 intron also increased GUS expression levels when fused in the correct orientation with the promoter of the unrelated COX5b-1 gene. Comparison of GUS activity values with the transcript levels suggests that the intron also increases translation efficiency of the corresponding mRNA. The results obtained point to an essential role of the intron present in the 5'-non-coding region of all known COX5c genes in directing the expression of these genes in plants.

5' Untranslated Regions↗

PubMed2004

Biochemical and functional analysis of a 9-nt RNA sequence that affects translation efficiency in eukaryotic cells.

We previously identified an internal ribosome entry site (IRES) within the 5' leader of the mRNA encoding the Gtx homeodomain protein and showed that shorter nonoverlapping segments of this 5' leader could enhance the translation of a second cistron in a dicistronic mRNA. One of these segments was 9 nt in length, and when multiple copies of this IRES module were linked together, IRES activity was greatly enhanced. To further expand the potential uses of these synthetic constructs and facilitate analyses of the mechanism by which they affect translation, we show here that an IRES containing five linked copies of the 9-nt sequence can also enhance translation in the 5' leader of a monocistronic mRNA. Moreover, a search for interactions of the IRES module with cellular factors revealed specific binding to 40S ribosomal subunits but not to other cellular components. Based on the results of earlier studies suggesting that this sequence could bind to a complementary segment of 18S rRNA, we tested various sequences for possible links between the length of the complementary match, their binding to ribosomes, and their influence on translational efficiency. We found that the length of the complementary match was directly correlated with the ability of RNA probes to bind to ribosomes. In addition, translation was maximally enhanced ( approximately 8-fold) by a 7-nt segment of the 9-nt element; the enhancement declined progressively as the complementary stretches became progressively longer or shorter. The results suggest that the Gtx 9-nt sequence affects translation efficiency by a mechanism that involves base pairing to 18S rRNA.

5' Untranslated Regions↗

PubMed1976

Individual translational efficiencies of SV40 and cellular mRNAs.

The technique of selective inhibition of peptide chain initiation by growth medium hypertonicity was adapted for cell monolayer cultures and applied to a study of protein synthesis in SV40 infected BSC-1 cells. The translational efficiences for individual peptide chain initiation sites on SV40 mRNAs were determined and compared to those for cellular mRNA species under conditions of a reduced rate of peptide chain initiation. The SV40 mRNAs show different translational efficiences. The results indicate that the synthesis of the SV40 proteins VP1, 2 and 3 are initiated independently and with different rates on viral mRNA(s). It is proposed, therefore, that VP2 is not a precursor for VP3.

Cell Line↗

PubMed1999

In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency

5' and 3' untranslated regions (UTRs) of plastid RNAs act as regulatory elements for post-transcriptional control of gene expression. Polyethylene glycol-mediated plastid transformation with UTR-GUS reporter gene fusions was used to study the function of the psbA, rbcL and rpl32 UTRs in vivo. All gene fusions were expressed from the same promoter, i.e. the promoter of the 16S-rRNA gene, such that variations in RNA and protein levels would be due to the involved UTR elements alone. Transgenic tobacco lines containing different combinations of UTRs showed fivefold variation in the uidA-mRNA level (RNA stability) and approximately 100-fold differences in GUS activity, a measure of translation activity. The rbcL 5'-UTR conferred greater mRNA stability than the psbA 5'-UTR on uidA transcripts. In contrast, the psbA 5'-UTR enhanced translation of GUS to a much greater extent compared to the rbcL 5'-UTR. The psbA 5'-UTR also mediated light-induced activation of translation which was not observed with other constructs. Deletion mutagenesis of an unanalysed terminal sequence element of the psbA 5'-UTR resulted in a twofold drop in uidA-mRNA level and a fourfold decrease in translation efficiency. Exchange of 3'-UTRs results in up to fivefold changes of mRNA levels and does not significantly influence translation efficiency. The mechanical impacts of these results on plastid translation regulation are discussed.

Journal Article↗

PubMed1994

Enhanced translational efficiency of a novel transforming growth factor beta 3 mRNA in human breast cancer cells.

The mRNA for transforming growth factor beta 3 (TGF-beta 3) includes a long (1.1-kb) 5' noncoding region which exerts a potent inhibitory effect on translational efficiency. We now report that many human breast cancer cell lines (T47-D, SK-BR-3, ZR-75-1, and BT-474) express two mRNA species for TGF-beta 3: the 3.5-kb transcript previously described as the only TGF-beta 3 mRNA species in cells and a novel 2.6-kb transcript which lacks approximately 870 nucleotides from the 5' noncoding region. The 5' end of the shorter transcript was sequenced, establishing it to be a 5' truncation of the full-length TGF-beta 3 transcript. Estradiol decreased mRNA levels of both TGF-beta 3 mRNA transcripts to an equivalent degree in estrogen receptor-positive cells. In contrast, the synthetic progestin gestodene altered the relative abundance of the two transcripts, preferentially diminishing the expression of the 2.6-kb transcript. The potential for enhanced mRNA translation attributable to the shorter 5' noncoding region was evaluated by transfection of cells with chimeric plasmid constructs in which the transcription unit consisted of coding sequence for chloramphenicol acetyltransferase downstream of the 5' noncoding sequence from TGF-beta 3. The translational efficiency of chloramphenicol acetyltransferase-encoding mRNA containing the shorter 5' noncoding region of the 2.6-kb TGF-beta 3 transcript was approximately seven times greater than with the full-length 5' noncoding region of TGF-beta 3. Polysome analysis of TGF-beta 3 mRNA in SK-BR-3 cells supported the hypothesis that the 2.6-kb transcript was more actively engaged in translation.

Base Sequence↗

PubMed1978

Protein synthesizing system from wheat germ: efficient translation of synthetic and natural messages.

Optimum conditions for translation of eukaryotic, prokaryotic and synthetic templates in wheat germ cell-free extract were determined. 1. Translation of eukaryotic message (BMV RNA and TMV RNA) was at optimum at the same concentrations of K+, Mg(2+), HEPES and spermine. In optimal conditions the efficiency of translation was high, for BMV RNA being equal to 220 pmoles and for TMV RNA, to 280 pmoles of leucine incorporated per 1 microgram of template. Prokaryotic template (Qbeta RNA) was translated under different ionic conditions. 2. Translation of synthetic template [poly(U)] was at optimum at fairly higher concentrations of K+ and Mg(2+) than those optimal for natural template translation. 3. Efficient translation of natural and synthetic templates depends on complete removal of inhibitors found in the wheat germ cell-free extract. Action of these inhibitors could be mimicked by adenine nucleotides.

Adenine Nucleotides↗

PubMed1995

Alternative leader sequences in insulin-like growth factor I mRNAs modulate translational efficiency and encode multiple signal peptides.

Rat insulin-like growth factor I (IGF-I) mRNAs contain multiple 5'-untranslated regions due to the use of leader exons transcribed from several transcription initiation sites and to alternative splicing within leader exon 1. Synthetic RNAs with 5'-ends corresponding to the use of exon 1 transcription initiation sites were translated in vitro into prepro-IGF-I peptides initiated at a Met-48 codon in exon 1 or a Met-22 codon in exon 3, and RNAs with a 5'-end corresponding to the major exon 2 transcription start site were translated into a prepro-IGF-I peptide initiated at a Met-32 codon in exon 2. All forms of prepro-IGF-I were processed by canine pancreatic microsomes, suggesting that all these prepeptides function as signal peptides. The translational efficiency of IGF-I RNAs was inversely proportional to the length of the 5'-untranslated region. Mutation of the first of three upstream AUG codons in exon 1, which potentially initiates a 14-amino acid open reading frame, did not affect prepro-IGF-I translation. The other two AUG codons are immediately followed by stop codons. The absence of both upstream AUG codons in a completely spliced exon 1-derived RNA enhanced the in vitro and in vivo translatability of this RNA as compared with the full-length RNA. Mutation of the downstream initiation codon in particular increased translational efficiency in vitro and in intact cells, suggesting that an inefficient reinitiation event at the Met-48 codon contributes to the poorer translation of IGF-I mRNAs in which these upstream AUGUGA motifs occur. We conclude that IGF-I mRNAs potentially encode multiple forms of preproIGF and that specific differences in their 5'-untranslated regions provide a molecular basis for translational control of IGF-I biosynthesis.

Animals↗

PubMed1999

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

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

Adenine↗

PubMed2004

Redefining the structure of the mouse connexin43 gene: selective promoter usage and alternative splicing mechanisms yield transcripts with different translational efficiencies.

The connexin43 (cx43) gene was originally described as consisting of two exons, one coding for most of the 5'-untranslated region (5'-UTR), and the other for the protein sequence and 3'-UTR. We now report that in mouse four additional exons are expressed, all coding for novel 5'-UTRs. Altogether, we found nine different cx43 mRNA species (GenBank accession numbers NM010288, and AY427554 through AY427561) generated by differential promoter usage and alternative splicing mechanisms. The relative abundance of these different mRNAs varied with the tissue source. In addition, the different transcripts showed varying translational efficiencies in several cell lines, indicating the presence of cis-RNA elements that regulate cx43 translation. We propose that it is the promoter driving the expression of the cx43 gene that determines exon choice in the downstream splicing events in a cell-type-dependent fashion. This in turn will affect the translation efficiency of the transcript orchestrating the events that lead to the final expression profile of cx43. Since a similar organization of the cx43 gene was also observed in rat it is likely that the complex regulation of cx43 expression involving transcription, splicing and translation mechanisms is a common trait conserved during evolution.

5' Untranslated Regions↗

PubMed2006

Improved translation efficiency of injected mRNA during early embryonic development.

Injection techniques are a powerful approach to study gene function in fish and frog model systems. In particular, in vitro transcribed mRNA is broadly used for such misexpression experiments. Sequence elements flanking the coding region, such as untranslated repeats and polyadenylation sequences, are known to affect the stability and the translation efficiency of mRNA. Here we show that in early embryos, poly(A) signals strongly contribute to the activity of the injected mRNA. Of interest, they only marginally affect mRNA stability, whereas the translation efficiency is dramatically enhanced. Combination of a poly(A) tail and an SV40 late poly(A) signal leads to highly synergistic effects of the two elements for injected mRNA. Compared with established vector systems, we detected a 20-fold improvement for mRNA derived from the novel transcription vector pMC.

Animals↗

PubMed2002

The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro.

Metazoan replication-dependent histone mRNAs end in a conserved stem-loop rather than in the poly(A) tail found on all other mRNAs. The 3' end of histone mRNA binds a single class of proteins, the stem-loop binding proteins (SLBP). In Xenopus, there are two SLBPs: xSLBP1, the homologue of the mammalian SLBP, which is required for processing of histone pre-mRNA, and xSLBP2, which is expressed only during oogenesis and is bound to the stored histone mRNA in Xenopus oocytes. The stem-loop is required for efficient translation of histone mRNAs and substitutes for the poly(A) tail, which is required for efficient translation of other eucaryotic mRNAs. When a rabbit reticulocyte lysate is programmed with uncapped luciferase mRNA ending in the histone stem-loop, there is a three- to sixfold increase in translation in the presence of xSLBP1 while xSLBP2 has no effect on translation. Neither SLBP affected the translation of a luciferase mRNA ending in a mutant stem-loop that does not bind SLBP. Capped luciferase mRNAs ending in the stem-loop were injected into Xenopus oocytes after overexpression of either xSLBP1 or xSLBP2. Overexpression of xSLBP1 in the oocytes stimulated translation, while overexpression of xSLBP2 reduced translation of the luciferase mRNA ending in the histone stem-loop. A small region in the N-terminal portion of xSLBP1 is required to stimulate translation both in vivo and in vitro. An MS2-human SLBP1 fusion protein can activate translation of a reporter mRNA ending in an MS2 binding site, indicating that xSLBP1 only needs to be recruited to the 3' end of the mRNA but does not need to be directly bound to the histone stem-loop to activate translation.

Animals↗

PubMed2002

Regulation of matrix metalloproteinase-9 (MMP-9) by translational efficiency in murine prostate carcinoma cells.

Expression of increased levels of matrix metalloproteinase-9 (MMP-9) has been implicated in tumor progression and angiogenesis. Much of our knowledge of the controls of MMP-9 levels has focused on transcription. Here we show that MMP-9 levels are also controlled by translational efficiency in murine prostate carcinoma cells. The murine prostate carcinoma cells 148-1,LMD and 148-1,PA were derived from a single mouse that had been implanted with urogenital sinus transformed by ras and myc. 148-1,PA secretes little MMP-9 yet has equivalent amounts of MMP-9 mRNA as the cell line 148-1,LMD that secretes substantially more. Infection with a retroviral vector for murine MMP-9 led to more expression of MMP-9 in both cases, but the differential remained. Human MMP-9 is equally expressed in both cells after infection with a vector for human MMP-9 indicating that the effect is species-specific. Pulse chase analysis revealed that MMP-9 was synthesized more rapidly in the 148-1,LMD cells than in the 148-1,PA cells. Markedly more MMP-9 mRNA was associated with polysomes in the cell line synthesizing more MMP-9. These results indicate regulation of MMP-9 synthesis at the level of translational efficiency.

Animals↗

PubMed1985

Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency.

The thymidine kinase gene of herpes simplex virus 1 was mutated by inserting oligodeoxynucleotide linkers into the region of the gene corresponding to the 5' untranslated portion of the mRNA. These linkers, when transcribed into mRNA, might be expected to form hairpin loops and hence to increase the secondary structure of the 5' end of the mRNA. Thymidine kinase insertion derivatives were examined in vivo and in vitro to determine translational efficiency. For the in vivo studies, thymidine kinase insertion derivatives were transfected into thymidine kinase deficient L cells alone and together with a selectable dominant marker, or were assayed in the COS-1 transient expression system. For in vitro studies, thymidine kinase insertion derivatives were subcloned into pSP64. Capped transcripts were analyzed for their ability to bind ribosomes and translate in rabbit reticulocyte lysates and wheat-germ extracts. The results demonstrate that translation efficiency is decreased as the number of linkers is increased and support the view that excessive secondary structure at the 5' end of eukaryotic mRNA impedes translation.

Animals↗

PubMed2003

Differential translational efficiency of ENaC subunits during lung development.

The amiloride-sensitive epithelial Na(+) channel (ENaC), the rate-limiting step in epithelial Na(+) transport, consists of three subunits: alpha, beta, and gamma. The abundance of mRNA encoding the alpha-subunit far surpasses the amount for other subunits, and considerably exceeds the predicted subunit protein stoichiometry. We evaluated 5'-untranslated region (UTR) expression and found that fetal rat lung uses alternative 5'UTRs for alpha-ENaC during development. Sucrose density gradient analysis of postnuclear supernatants from fetal rat lung homogenates demonstrated that all three ENaC subunits were associated with high molecular weight polysomes, indicating active translation of the mRNAs, but translational efficiency was much lower for the alpha-subunit. Sucrose density gradient distributions were comparable for the endogenously expressed alpha-ENaC 5'UTRs in rat lung at Fetal Day 20 or Postnatal Day 1 using Northern analysis. Although birth resulted in a global decrease in lung mRNA translation, the loading of ribosomes on ENaC subunit mRNAs was largely unaffected. Evaluation of cytokeratin 18 and vimentin mRNAs in these gradients suggested a cell-specific effect. We conclude that there are different translational efficiencies for ENaC subunits and that perinatal processes globally modulate lung mRNA translation.

5' Untranslated Regions↗

PubMed1999

Modulation of translational efficiency by contextual nucleotides flanking a baculovirus initiator AUG codon.

In a previous study of translational regulation of a baculovirus gene, we observed that translation initiated at an unexpectedly high efficiency from an AUG codon found in what was believed to be a poor context (M.-J. Chang and G. W. Blissard, 1997, J. Virol. 71, 7448-7460). In the current study, we examined the roles of nucleotides flanking a baculovirus AUG initiator codon in modulating translation initiation in lepidopteran insect cells. The roles of nucleotides flanking the AcMNPV gp64 initiator codon were examined by site-directed mutagenesis and functional assays in transfected Sf9 cells. To eliminate potential cis-acting sequences and effects, the gp64 initiator context was cloned in-frame with a chloramphenicol acetyl transferase reporter gene and under the control of a heterologous promoter. All possible single-nucleotide substitutions were generated in positions -6 to -1 and +4 to +6, relative to the A of the initiator AUG codon, which was designated +1. Constructs were transfected into lepidopteran cells and translation products were quantified by an enzyme-linked immunosorbent assay procedure. Substitutions of pyrimidines or other nucleotides at the -3 position resulted in little or no detectable effect on translation efficiency. In contrast, specific substitutions at the +4 and +5 positions resulted in approximately 2- to 3-fold increases in translation. Substitution of A in the +4 position resulted in an approximately 3-fold increase in translation, and substitution of any nucleotide for T in the +5 position resulted in approximately 1.9- to 2.8-fold increases. Substitutions at other positions (-6 to -1 and +6) resulted in no detectable increase or decrease in translation efficiency. These experimental results suggest an optimal initiator context of 5'-N N N N N N A U G A a/c/g N-3' for efficient translation initiation in lepidopteran cells. Consensus translation initiation contexts were generated from baculovirus genes and lepidopteran genes, then compared with the experimental results from the gp64 initiator context.

Animals↗

PubMed1988

Beta-globin mRNAs capped with m7G, m2.7(2)G or m2.2.7(3)G differ in intrinsic translation efficiency.

We examined the intramolecular effect of altered cap structures on translation efficiency of artificial beta-globin mRNAs. For these studies, synthetic dinucleotides of the form X(5')ppp(5')G [X = 7-methyl guanosine (m7G), 2,7-dimethyl guanosine (m2(2,7)G) or 2,2,7-trimethyl guanosine (m3(2,2,7)G)], were transcriptionally incorporated into mRNAs, containing rabbit beta-globin coding sequences, using T7 RNA polymerase and a beta-globin cDNA template. These synthetic mRNAs were assayed in reticulocyte lysate for activity relative to m7G-capped mRNA. m2(2,7)G-Capped mRNA was found to be 1.5-fold more active than m7G-capped mRNA. Messenger RNA capped with m3(2,2,7)G was less active with activity of 0.24 relative to its m7G-capped counterpart (activity = 1.0). These data suggest that m7G-capped mRNAs become more active as translation templates after addition of a single N2 methyl moiety, which is especially pertinent to gene expression in togaviridae. The latter are observed to synthesize m2(2,7)G and m3(2,2,7)G-capped mRNAs in addition to m7G-capped templates during the course of infection in animal cells.

Animals↗

PubMed1988

Mutations in the leader sequence and initiation codon of the gene for ribosomal protein S20 (rpsT) affect both translational efficiency and autoregulation.

We have transferred the complete structural gene and part of the leader for ribosomal protein S20 of Escherichia coli to a controllable expression vector and have used oligonucleotide-directed mutagenesis to create mutations in the untranslated leader of the plasmid-borne gene. We have assayed for posttranscriptional regulation of the synthesis of S20 after inducing transcription of the mutant S20 mRNA from the expression vector. We found that two mutations lead to loss of feedback control of S20 synthesis: (i) a change of the initiation codon from UUG to AUG and (ii) a replacement of part of the S20 leader with a nonhomologous sequence including an AUG initiation codon. These mutations also lead to increases in both the intrinsic translational efficiency of the plasmid-encoded S20 mRNA in vitro and its half-life in vivo. A double mutation (GA to CT) at residues -3 and -4 relative to the initiation codon does not result in overproduction of S20. Rather, it reduces translational efficiency in vitro and mRNA stability in vivo. Our results demonstrate the fundamental importance of the UUG initiation codon in mediating autogenous repression of S20 synthesis.

Base Sequence↗