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

A firefly luciferase (FLuc) counts among the most popular reporters of present-day molecular and cellular biology. In this study, we report a cryptic promoter activity in the luc+ gene, which is the most frequently used version of the firefly luciferase. The FLuc coding region displays cryptic promoter activity both in mammalian and yeast cells. In human CCL13 and Huh7 cells, cryptic transcription from the luc+ gene is 10-16 times weaker in comparison to the strong immediate-early cytomegalovirus promoter. Additionally, we discuss a possible impact of the FLuc gene cryptic promoter on experimental results especially in some fields of the RNA-oriented research, for example, in analysis of translation initiation or analysis of miRNA/siRNA function. Specifically, we propose how this newly described cryptic promoter activity within the FLuc gene might contribute to the previous determination of the strength of the cryptic promoter found in the cDNA corresponding to the hepatitis C virus internal ribosome entry site. Our findings should appeal to the researchers to be more careful when designing firefly luciferase-based assays as well as open the possibility of performing some experiments with the hepatitis C virus internal ribosome entry site, which could not be considered until now.

• MeSH
• buněčné linie MeSH
• genetická transkripce * MeSH
• Hepacivirus genetika   MeSH
• komplementární DNA genetika   MeSH
• lidé MeSH
• luciferasy světlušek genetika   MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• komplementární DNA MeSH
• luciferasy světlušek MeSH