Transfer RNAs (tRNAs) serve as a dictionary for the ribosome translating the genetic message from mRNA into a polypeptide chain. In addition to this canonical role, tRNAs are involved in other processes such as programmed stop codon readthrough (SC-RT). There, tRNAs with near-cognate anticodons to stop codons must outcompete release factors and incorporate into the ribosomal decoding center to prevent termination and allow translation to continue. However, not all near-cognate tRNAs promote efficient SC-RT. Here, with the help of Saccharomyces cerevisiae and Trypanosoma brucei, we demonstrate that those tRNAs that promote efficient SC-RT establish critical contacts between their anticodon stem (AS) and ribosomal proteins Rps30/eS30 and Rps25/eS25 forming the decoding site. Unexpectedly, the length and well-defined nature of the AS determine the strength of these contacts, which is reflected in organisms with reassigned stop codons. These findings open an unexplored direction in tRNA biology that should facilitate the design of artificial tRNAs with specifically altered decoding abilities.

• MeSH
• antikodon metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• proteosyntéza * MeSH
• ribozomální proteiny metabolismus   MeSH
• ribozomy * metabolismus   MeSH
• RNA transferová * metabolismus   genetika   chemie   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• terminační kodon * genetika   metabolismus   MeSH
• Trypanosoma brucei brucei genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antikodon MeSH
• ribozomální proteiny MeSH
• RNA transferová * MeSH
• terminační kodon * MeSH

Dual reporters encoding two distinct proteins within the same mRNA have had a crucial role in identifying and characterizing unconventional mechanisms of eukaryotic translation. These mechanisms include initiation via internal ribosomal entry sites (IRESs), ribosomal frameshifting, stop codon readthrough and reinitiation. This design enables the expression of one reporter to be influenced by the specific mechanism under investigation, while the other reporter serves as an internal control. However, challenges arise when intervening test sequences are placed between these two reporters. Such sequences can inadvertently impact the expression or function of either reporter, independent of translation-related changes, potentially biasing the results. These effects may occur due to cryptic regulatory elements inducing or affecting transcription initiation, splicing, polyadenylation and antisense transcription as well as unpredictable effects of the translated test sequences on the stability and activity of the reporters. Unfortunately, these unintended effects may lead to misinterpretation of data and the publication of incorrect conclusions in the scientific literature. To address this issue and to assist the scientific community in accurately interpreting dual-reporter experiments, we have developed comprehensive guidelines. These guidelines cover experimental design, interpretation and the minimal requirements for reporting results. They are designed to aid researchers conducting these experiments as well as reviewers, editors and other investigators who seek to evaluate published data.

• MeSH
• Eukaryota genetika   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• proteosyntéza genetika   MeSH
• reportérové geny * MeSH
• směrnice jako téma MeSH
• výzkumný projekt normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• messenger RNA MeSH

Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation (REI) at short upstream open reading frames (uORFs) harboring penultimate codons that confer heightened dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited REI at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on REI at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) REI. We found that the Tma proteins generally impede REI at native uORF4 and its variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on REI at native uORF1 and equipping it with Tma-hyperdependent penultimate codons generally did not confer Tma-dependent REI; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the REI potential of the uORF and penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

• MeSH
• iniciace translace peptidového řetězce MeSH
• malé podjednotky ribozomu eukaryotické * metabolismus   genetika   MeSH
• messenger RNA * metabolismus   genetika   MeSH
• otevřené čtecí rámce * MeSH
• proteosyntéza MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   genetika   MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• transkripční faktory bZIP * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• messenger RNA * MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• transkripční faktory bZIP * MeSH

Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S subunit from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation at short upstream open reading frames (uORFs) harboring penultimate codons that confer dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited reinitiation at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on reinitiation at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) reinitiation. We found that the Tma proteins generally impede reinitiation at native uORF4 and uORF4 variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on reinitiation at native uORF1, and equipping uORF1 with Tma-dependent penultimate codons generally did not confer Tma-dependent reinitiation; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the reinitiation potential of the uORF and the penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

• Klíčová slova
• DENR, MCTS1, Tma, eIF2D, reinitiation, ribosome recycling,
• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

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.

• Klíčová slova
• cysteine tRNA, near-cognate tRNA, readthrough-inducing tRNA, stop codon readthrough, translation,
• 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
• Názvy látek
• antikodon MeSH
• cystein * MeSH
• nesmyslný kodon MeSH
• RNA transferová Cys MeSH
• RNA transferová Trp MeSH
• RNA transferová Tyr MeSH
• RNA transferová MeSH
• terminační kodon MeSH

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.

• MeSH
• antikodon * chemie   genetika   metabolismus   MeSH
• Ciliophora genetika   MeSH
• eukaryotické buňky * MeSH
• genetický kód * genetika   MeSH
• mutace * MeSH
• peptidy - faktory ukončení * genetika   metabolismus   MeSH
• RNA transferová Glu genetika   MeSH
• RNA transferová Trp genetika   MeSH
• RNA transferová * genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• terminační kodon * genetika   MeSH
• Trypanosoma brucei brucei genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antikodon * MeSH
• peptidy - faktory ukončení * MeSH
• RNA transferová Glu MeSH
• RNA transferová Trp MeSH
• RNA transferová * MeSH
• terminační kodon * MeSH

Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.

• MeSH
• buněčné linie MeSH
• lidé MeSH
• mutace MeSH
• nádorový supresorový protein p53 biosyntéza   genetika   MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) MeSH
• proteiny genetika   MeSH
• proteosyntéza * MeSH
• reportérové geny MeSH
• RNA malá jaderná genetika   MeSH
• RNA transferová Trp genetika   metabolismus   MeSH
• RNA transferová Tyr genetika   metabolismus   MeSH
• terminační kodon * MeSH
• tryptofan-tRNA-ligasa genetika   MeSH
• tyrosin-tRNA-ligasa genetika   MeSH
• virové proteiny genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorový supresorový protein p53 MeSH
• proteiny MeSH
• RNA malá jaderná MeSH
• RNA transferová Trp MeSH
• RNA transferová Tyr MeSH
• terminační kodon * MeSH
• tryptofan-tRNA-ligasa MeSH
• tyrosin-tRNA-ligasa MeSH
• U6 small nuclear RNA MeSH Prohlížeč
• virové proteiny 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
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• ribozomální proteiny MeSH
• RNA transferová MeSH
• RPS3 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• terminační kodon 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
• Názvy látek
• eukaryotický iniciační faktor 1 MeSH
• eukaryotický iniciační faktor 3 MeSH
• eukaryotický iniciační faktor 5 MeSH
• Saccharomyces cerevisiae - proteiny 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
• Názvy látek
• aminoglykosidy MeSH
• nukleotidy MeSH
• RNA transferová Gln MeSH
• RNA transferová Tyr MeSH
• RNA transferová MeSH
• terminační kodon * MeSH