The upstream open reading frame of cyclin-dependent kinase inhibitor 1A mRNA negatively regulates translation of the downstream main open reading frame

The skeletal muscle cells are one of the main sites of glucose uptake through glucose transporter 4 (GLUT4) in response to insulin. In muscle cells, 5' adenosine monophosphate-activated protein kinase (AMPK) is known as another GLUT4 translocation promoter. Natural compounds that activate AMPK have a possibility to overcome insulin resistance in the diabetic state. Piceatannol is a natural analog and a metabolite of resveratrol, a known AMPK activator. In this study, we investigate the in vitro effect of piceatannol on glucose uptake, AMPK phosphorylation and GLUT4 translocation to plasma membrane in L6 myocytes, and its in vivo effect on blood glucose levels in type 2 diabetic model db/db mice. Piceatannol was found to promote glucose uptake, AMPK phosphorylation and GLUT4 translocation by Western blotting analyses in L6 myotubes under a condition of insulin absence. Promotion by piceatannol of glucose uptake as well as GLUT4 translocation to plasma membrane by immunocytochemistry was also demonstrated in L6 myoblasts transfected with a glut4 cDNA-coding vector. Piceatannol suppressed the rises in blood glucose levels at early stages and improved the impaired glucose tolerance at late stages in db/db mice. These in vitro and in vivo findings suggest that piceatannol may be preventive and remedial for type 2 diabetes and become an antidiabetic phytochemical.     

Maintenance of the correct open reading frame by the ribosome

During translation, a string of non-overlapping triplet codons in messenger RNA is decoded into protein. The ability of a ribosome to decode mRNA without shifting between reading frames is a strict requirement for accurate protein biosynthesis. Despite enormous progress in understanding the mechanism of transfer RNA selection, the mechanism by which the correct reading frame is maintained remains unclear. In this report, evidence is presented that supports the idea that the translational frame is controlled mainly by the stability of codon–anticodon interactions at the P site. The relative instability of such interactions may lead to dissociation of the P-site tRNA from its codon, and formation of a complex with an overlapping codon, the process known as P-site tRNA slippage. We propose that this process is central to all known cases of +1 ribosomal frameshifting, including that required for the decoding of the yeast transposable element Ty3. An earlier model for the decoding of this element proposed 'out-of-frame' binding of A-site tRNA without preceding P-site tRNA slippage.     

Binding of ribosome recycling factor to ribosomes,comparison with tRNA

The prokaryotic post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Because of the structural similarity of RRF and tRNA, we compared the biochemical characteristics of RRF binding to ribosomes with that of tRNA. Unesterified tRNA inhibited the disassembly of the post-termination complex in a competitive manner with RRF, suggesting that RRF binds to the A-site. Approximately one molecule of ribosome-bound RRF was detected after isolation of the RRF-ribosome complex. RRF and unesterified tRNA similarly inhibited the binding of N-acetylphenylalanyl-tRNA to the P-site of non-programmed but not programmed ribosomes. Under the conditions in which unesterified tRNA binds to both the P- and E-sites of non-programmed ribosomes, RRF inhibited 50% of the tRNA binding, suggesting that RRF does not bind to the E-site. The results are consistent with the notion that a single RRF binds to the A- and P-sites in a somewhat analogous manner to the A/P-site bound peptidyl tRNA. The binding of RRF and tRNA to ribosomes was influenced by Mg(2+) and NH(4)(+) ions in a similar manner.     

In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from the S-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism.     

Fast recycling of Escherichia coli ribosomes requires both ribosome recycling factor (RRF) and release factor RF3.

A complete translation system has been assembled from pure initiation, elongation and termination factors as well as pure aminoacyl-tRNA synthetases. In this system, ribosomes perform repeated rounds of translation of short synthetic mRNAs which allows the time per translational round (the recycling time) to be measured. The system has been used to study the influence of release factor RF3 and of ribosome recycling factor RRF on the rate of recycling of ribosomes. In the absence of both RF3 and RRF, the recycling time is approximately 40 s. This time is reduced to approximately 30 s by the addition of RF3 alone and to approximately 15 s by the addition of RRF alone. When both RF3 and RRF are added to the translation system, the recycling time drops to <6 s. Release factor RF3 is seen to promote RF1 cycling between different ribosomes. The action of RRF is shown to depend on the concentration of elongation factor-G. Even in the presence of RRF, ribosomes do not leave the mRNA after termination, but translate the same mRNA several times. This shows that RRF does not actively eject mRNA from the terminating ribosome. It is proposed that terminating ribosomes become mobile on mRNA and ready to enter the next translation round only after two distinct steps, catalysed consecutively by RF3 and RRF, which are slow in the absence of these factors.     

The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The three-factor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.     

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.     

Coupled termination/reinitiation for translation of the downstream open reading frame B of the prototypic hypovirus CHV1-EP713

Cryphonectria hypovirus 1 (CHV1), associated with the picorna-like superfamily, infects the chestnut blight fungus and attenuates the virulence of the host fungus. The genomic RNA of the virus has two continuous open reading frames, A and B, separated by the pentanucleotide UAAUG. We present here evidence suggesting that ORF B is translated from genome-sized virus mRNA by a coupled termination/reinitiation mechanism mediated by the pentamer. In the coupled translation, the overlapping UAA and AUG triplets serve as the stop codon of ORF A and the initiator of ORF B, respectively. This was established by the use of a luciferase assay with a basic construct containing the ORF A sequence and the firefly luciferase gene while retaining the pentamer between the two coding sequences. The proportion of ribosomes reinitiating translation after terminating was determined to be 2.5–4.4% by three independent assay systems in fungal and insect cells. Use of a series of mutant constructs identified two sequence elements, the pentamer and the p40 sequence, that affect the efficiency of coupled translation and virus replication. Together, these results provide the first example of coupled translation facilitated by the pentanucleotide UAAUG in the kingdom Fungi. The mechanism by which the preceding p40-coding sequence promotes reinitiation is discussed.     

Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase

Synthesis of S-adenosylmethionine decarboxylase (AdoMetDC), a key regulated enzyme in the pathway of polyamine biosynthesis, is feedback-controlled at the level of translation by spermidine and spermine. The peptide product of an upstream open reading frame (uORF) in the mRNA is solely responsible for polyamine regulation of AdoMetDC translation. Using a primer extension inhibition assay and in vitro protein synthesis reactions, we found ribosomes paused at or close to the termination codon of the uORF. This pause was greatly diminished with the altered uORFs' sequences that abolish uORF regulation in vivo. The half-life of the ribosome pause was related to the concentration of polyamines present but was unaffected by magnesium concentration. Furthermore, inhibition of translation initiation at a reporter gene placed downstream of the AdoMetDC uORF directly correlated with the stability of the ribosome pause at the uORF. These observations are consistent with a model in which regulation of ribosome pausing at the uORF by polyamines controls ribosome access to the downstream AdoMetDC reading frame.     

Specific binding of ribosome recycling factor (RRF) with the Escherichia coli ribosomes by BIACORE

The direct assays on Biacore with immobilised RRF and purified L11 from E. coli in the flow trough have shown unspecific binding between the both proteins. The interaction of RRF with GTPase domain of E. coli ribosomes, a functionally active complex of L11 with 23S r RNA and L10.(L7/L12)₄ was studied by Biacore. In the experiments of binding of RRF with 30S, 50S and 70S ribosomes from E. coli were used the antibiotics thiostrepton, tetracycline and neomycin and factors, influencing the 70S dissociation Mg²⁺, NH₄Cl, EDTA. The binding is strongly dependent from the concentrations of RRF, Mg²⁺, NH₄Cl, EDTA and is inhibited by thiostrepton. The effect is most specific for 50S subunits and indicates that the GTPase centre can be considered as a possible site of interaction of RRF with the ribosome. We can consider an electrostatic character of the interactions with most probable candidate 16S and 23S r RNA at the interface of 30S and 50S ribosomal subunits.     

Inhibitory effect of heterologous ribosome recycling factor on growth of Escherichia coli

Ribosome recycling factor (RRF) of Thermotoga maritima was expressed in Escherichia coli from the cloned T. maritima RRF gene and purified. Expression of T. maritima RRF inhibited growth of the E. coli host in a dose-dependent manner, an effect counteracted by the overexpression of E. coli RRF. T. maritima RRF also inhibited the E. coli RRF reaction in vitro. Genes encoding RRFs from Streptococcus pneumoniae and Helicobacter pylori have been cloned, and they also impair growth of E. coli, although the inhibitory effect of these RRFs was less pronounced than that of T. maritima RRF. The amino acid sequence at positions 57 to 62, 74 to 78, 118 to 122, 154 to 160, and 172 to 176 in T. maritima RRF differed totally from that of E. coli RRF. This suggests that these regions are important for the inhibitory effect of heterologous RRF. We further suggest that bending and stretching of the RRF molecule at the hinge between two domains may be critical for RRF activity and therefore responsible for T. maritima RRF inhibition of the E. coli RRF reaction.     

In vitro and in vivo characterization of a murine cytomegalovirus with a mutation at open reading frame m166

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the mutants, Rvm166, which contained the transposon sequence at open reading frame m166, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. The viral mutant replicated as well as the wild-type Smith strain in vitro in NIH 3T3 cells, whereas the transposon insertion precluded the expression of >65% of the m166 open reading frame. Compared to the wild-type strain and a rescued virus that restored the m166 region, the viral mutant was significantly attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. At 21 days postinfection, the titers of the viral mutant in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice were lower than the titers of the Smith strain and the rescued virus by about 30000-, 10000-, 1000-, 300-, and 800-fold, respectively. Moreover, the virulence of the mutant virus appears to be severely attenuated because no death was found in SCID mice infected with the viral mutant up to 90 days postinfection, whereas all of the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results suggest that m166 probably encodes a virulence factor and is required for MCMV virulence in killing SCID mice and for optimal viral growth in vivo.