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

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