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
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
One of the extensively studied mechanisms of gene-specific translational regulation is reinitiation. It takes place on messenger RNAs (mRNAs) where main ORF is preceded by upstream ORF (uORF). Even though uORFs generally down-regulate main ORF expression, specific uORFs exist that allow high level of downstream ORF expression. The key is their ability to retain 40S subunits on mRNA upon termination of their translation to resume scanning for the next AUG. Here, we took advantage of the exemplary model system of reinitiation, the mRNA of yeast transcriptional activator GCN4 containing four short uORFs, and show that contrary to previous reports, not only the first but the first two of its uORFs allow efficient reinitiation. Strikingly, we demonstrate that they utilize a similar molecular mechanism relying on several cis-acting 5' reinitiation-promoting elements, one of which they share, and the interaction with the a/TIF32 subunit of translation initiation factor eIF3. Since a similar mechanism operates also on YAP1 uORF, our findings strongly suggest that basic principles of reinitiation are conserved. Furthermore, presence of two consecutive reinitiation-permissive uORFs followed by two reinitiation-non-permissive uORFs suggests that tightness of GCN4 translational control is ensured by a fail-safe mechanism that effectively prevents or triggers GCN4 expression under nutrient replete or deplete conditions, respectively.
- MeSH
- 5' nepřekládaná oblast MeSH
- eukaryotický iniciační faktor 3 metabolismus MeSH
- fungální RNA genetika metabolismus MeSH
- iniciace translace peptidového řetězce * MeSH
- messenger RNA genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- otevřené čtecí rámce MeSH
- regulace genové exprese u hub MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- transkripční faktory bZIP genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
