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

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