Under certain circumstances, any of the three termination codons can be read through by a near-cognate tRNA; i.e., a tRNA whose two out of three anticodon nucleotides base pair with those of the stop codon. Unless programed to synthetize C-terminally extended protein variants with expanded physiological roles, readthrough represents an undesirable translational error. On the other side of a coin, a significant number of human genetic diseases is associated with the introduction of nonsense mutations (premature termination codons [PTCs]) into coding sequences, where stopping is not desirable. Here, the tRNA's ability to induce readthrough opens up the intriguing possibility of mitigating the deleterious effects of PTCs on human health. In yeast, the UGA and UAR stop codons were described to be read through by four readthrough-inducing rti-tRNAs-tRNATrp and tRNACys, and tRNATyr and tRNAGln, respectively. The readthrough-inducing potential of tRNATrp and tRNATyr was also observed in human cell lines. Here, we investigated the readthrough-inducing potential of human tRNACys in the HEK293T cell line. The tRNACys family consists of two isoacceptors, one with ACA and the other with GCA anticodons. We selected nine representative tRNACys isodecoders (differing in primary sequence and expression level) and tested them using dual luciferase reporter assays. We found that at least two tRNACys can significantly elevate UGA readthrough when overexpressed. This indicates a mechanistically conserved nature of rti-tRNAs between yeast and human, supporting the idea that they could be used in the PTC-associated RNA therapies.
- MeSH
- antikodon MeSH
- cystein * genetika metabolismus MeSH
- HEK293 buňky MeSH
- lidé MeSH
- nesmyslný kodon genetika MeSH
- proteosyntéza MeSH
- RNA transferová Cys metabolismus MeSH
- RNA transferová Trp metabolismus MeSH
- RNA transferová Tyr MeSH
- RNA transferová genetika metabolismus MeSH
- Saccharomyces cerevisiae * genetika MeSH
- terminační kodon genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
One of the key roles of the 12-subunit eukaryotic translation initiation factor 3 (eIF3) is to promote the formation of the 43S and 48S pre-initiation complexes (PICs). However, particular contributions of its individual subunits to these two critical initiation reactions remained obscure. Here, we adapted formaldehyde gradient cross-linking protocol to translation studies and investigated the efficiency of the 43S and 48S PIC assembly in knockdowns of individual subunits of human eIF3 known to produce various partial subcomplexes. We revealed that eIF3d constitutes an important intermolecular bridge between eIF3 and the 40S subunit as its elimination from the eIF3 holocomplex severely compromised the 43S PIC assembly. Similarly, subunits eIF3a, c and e were found to represent an important binding force driving eIF3 binding to the 40S subunit. In addition, we demonstrated that eIF3c, and eIF3k and l subunits alter the efficiency of mRNA recruitment to 43S PICs in an opposite manner. Whereas the eIF3c knockdown reduces it, downregulation of eIF3k or eIF3l increases mRNA recruitment, suggesting that the latter subunits possess a regulatory potential. Altogether this study provides new insights into the role of human eIF3 in the initial assembly steps of the translational machinery.
- MeSH
- eukaryotický iniciační faktor 3 genetika MeSH
- formaldehyd farmakologie MeSH
- lidé MeSH
- malé podjednotky ribozomu eukaryotické genetika MeSH
- messenger RNA genetika MeSH
- proteiny asociované s mikrotubuly genetika MeSH
- proteosyntéza genetika MeSH
- reagencia zkříženě vázaná farmakologie MeSH
- ribozomy genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.
- MeSH
- eukaryotický iniciační faktor 3 genetika metabolismus MeSH
- geneticky modifikované organismy MeSH
- proteosyntéza genetika MeSH
- ribozomální proteiny genetika fyziologie MeSH
- ribozomy metabolismus MeSH
- RNA transferová metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika fyziologie MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- terminace translace peptidového řetězce * genetika MeSH
- terminační kodon metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
eIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.
- MeSH
- elektronová kryomikroskopie MeSH
- eukaryotický iniciační faktor 1 chemie genetika metabolismus MeSH
- eukaryotický iniciační faktor 3 chemie genetika metabolismus MeSH
- eukaryotický iniciační faktor 5 chemie genetika metabolismus MeSH
- iniciace translace peptidového řetězce * MeSH
- malé podjednotky ribozomu eukaryotické genetika metabolismus MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus ultrastruktura MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Stop codon readthrough-the decoding of a stop codon by a near-cognate tRNA-is employed by viruses to balance levels of enzymatic and structural proteins and by eukaryotic cells to enable isoform-specific protein synthesis in response to external stimuli. Owing to the prevalence of premature termination codons in human disease, readthrough has emerged as an attractive therapeutic target. A growing list of various features, for example the +4 nucleotide immediately following the stop codon, modulate readthrough levels, underscoring the need for systematic investigation of readthrough. Here, we identified and described a complete group of yeast tRNAs that induce readthrough in the stop-codon tetranucleotide manner when overexpressed, designated readthrough-inducing tRNAs (rti-tRNAs). These rti-tRNAs are the keystones of YARIS (yeast applied readthrough inducing system), a reporter-based assay enabling simultaneous detection of readthrough levels at all twelve stop-codon tetranucleotides and as a function of the complete set of rti-tRNAs. We demonstrate the utility of YARIS for systematic study of translation readthrough by employing it to interrogate the effects of natural rti-tRNA modifications, as well as various readthrough-inducing drugs (RTIDs). This analysis identified a variety of genetic interactions demonstrating the power of YARIS to characterize existing and identify novel RTIDs.
- MeSH
- aminoglykosidy farmakologie MeSH
- nukleotidy chemie MeSH
- proteosyntéza * účinky léků MeSH
- RNA transferová Gln MeSH
- RNA transferová Tyr MeSH
- RNA transferová metabolismus MeSH
- Saccharomyces cerevisiae genetika MeSH
- terminační kodon * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ultra-high molecular polyethylene (UHMWPE) is one of the most used materials of the acetabular liners in total tip arthroplasty (THA). Polyethylene has good tribological properties and biocompatibility. However, the lifetime of polyethylene implants is limited by wear related complications. Polyethylene material released into the periprosthetic environment induces osteolysis that can be followed by implant loosening. Wear of cup is influenced mainly by orientation of the cup in pelvis, by initial geometry before the material degradation and by tribological parameters. Aim of this study is to focus on the run-in-phase of the liner which is predictive for future life cycles of liner. Creep deformations of liners for 30°, 45°, 60° inclination angles surgically recommended for the positioning in pelvis were analyzed by the optical scanning method. Load tests were performed for 50,000 cycles. Creep deformations and surface changes were analyzed at each 10,000 cycles. The results showed that liners with 60° inclination angle had higher creep deformations. Penetration of femoral head was 0.04-0.05 mm and occupied bearing area was around 77%. The smallest creep was measured for the 45° angle. However, deformation in the superior quadrant of acetabular rim, which is vulnerable for potential fracture of a liner, was identified in this case. Topography of the surface bearing was also observed during the run-in-phase. The surface was smoothened and showed multidirectional scratches caused by the influence of third body particles. This phase was followed by early delamination. Flakes sized approximately 5-20 µm were observed on the UHMWPE surface. This is similar to the'flake' shape wear debris extracted in vivo. Detailed analysis of run-in phase of loading of modern polyethylene implants can help to distinguish between their creep deformation and true degradation. The latter contributes strongly to the development of wear related complications associated with THAs limiting substantially their time in service.
Translation reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream ORFs to prevent recycling of the post-termination 40S subunit in order to resume scanning for reinitiation downstream. Its efficiency decreases with the increasing uORF length, or by the presence of secondary structures, suggesting that the time taken to translate a uORF is more critical than its length. This led to a hypothesis that some initiation factors needed for reinitiation are preserved on the 80S ribosome during early elongation. Here, using the GCN4 mRNA containing four short uORFs, we developed a novel in vivo RNA-protein Ni2+-pull down assay to demonstrate for the first time that one of these initiation factors is eIF3. eIF3 but not eIF2 preferentially associates with RNA segments encompassing two GCN4 reinitiation-permissive uORFs, uORF1 and uORF2, containing cis-acting 5΄ reinitiation-promoting elements (RPEs). We show that the preferred association of eIF3 with these uORFs is dependent on intact RPEs and the eIF3a/TIF32 subunit and sharply declines with the extended length of uORFs. Our data thus imply that eIF3 travels with early elongating ribosomes and that the RPEs interact with eIF3 in order to stabilize the mRNA-eIF3-40S post-termination complex to stimulate efficient reinitiation downstream.
- MeSH
- 5' nepřekládaná oblast MeSH
- elongace translace peptidového řetězce MeSH
- eukaryotický iniciační faktor 3 metabolismus MeSH
- genetické techniky MeSH
- iniciace translace peptidového řetězce * MeSH
- malé podjednotky ribozomu eukaryotické metabolismus MeSH
- otevřené čtecí rámce * MeSH
- regulace genové exprese * MeSH
- ribozomy metabolismus MeSH
- terminace translace peptidového řetězce MeSH
- terminační kodon MeSH
- Publikační typ
- časopisecké články MeSH
Reinitiation after translation of short upstream ORFs (uORFs) represents one of the means of regulation of gene expression on the mRNA-specific level in response to changing environmental conditions. Over the years it has been shown-mainly in budding yeast-that its efficiency depends on cis-acting features occurring in sequences flanking reinitiation-permissive uORFs, the nature of their coding sequences, as well as protein factors acting in trans. We earlier demonstrated that the first two uORFs from the reinitiation-regulated yeast GCN4 mRNA leader carry specific structural elements in their 5' sequences that interact with the translation initiation factor eIF3 to prevent full ribosomal recycling post their translation. Actually, this interaction turned out to be instrumental in stabilizing the mRNA·40S post-termination complex, which is thus capable to eventually resume scanning and reinitiate on the next AUG start site downstream. Recently, we also provided important in vivo evidence strongly supporting the long-standing idea that to stimulate reinitiation, eIF3 has to remain bound to ribosomes elongating these uORFs until their stop codon has been reached. Here we examined the importance of eIF3 and sequences flanking uORF1 of the human functional homolog of yeast GCN4, ATF4, in stimulation of efficient reinitiation. We revealed that the molecular basis of the reinitiation mechanism is conserved between yeasts and humans.
- MeSH
- eukaryotický iniciační faktor 3 chemie metabolismus MeSH
- iniciace translace peptidového řetězce * MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- otevřené čtecí rámce * MeSH
- proteosyntéza MeSH
- ribozomy metabolismus MeSH
- savci MeSH
- transkripční faktor ATF4 chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Protein synthesis is mediated via numerous molecules including the ribosome, mRNA, tRNAs, as well as translation initiation, elongation and release factors. Some of these factors play several roles throughout the entire process to ensure proper assembly of the preinitiation complex on the right mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most intriguing of these multitasking factors is the eukaryotic initiation factor eIF3. Recent evidence strongly suggests that this factor, which coordinates the progress of most of the initiation steps, does not come off the initiation complex upon subunit joining, but instead it remains bound to 80S ribosomes and gradually falls off during the first few elongation cycles to: (1) promote resumption of scanning on the same mRNA molecule for reinitiation downstream-in case of translation of upstream ORFs short enough to preserve eIF3 bound; or (2) come back during termination on long ORFs to fine tune its fidelity or, if signaled, promote programmed stop codon readthrough. Here, we unite recent structural views of the eIF3-40S complex and discus all known eIF3 roles to provide a broad picture of the eIF3's impact on translational control in eukaryotic cells.
- MeSH
- eukaryotický iniciační faktor 3 chemie genetika metabolismus MeSH
- konformace proteinů * MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- molekulární modely MeSH
- podjednotky proteinů chemie genetika metabolismus MeSH
- proteosyntéza * MeSH
- ribozomy genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
