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.

Belozersky Institute of Physico Chemical Biology Lomonosov Moscow State University Moscow Russia

Center for Biomolecular Sciences University of Illinois Chicago Chicago IL USA

Departament de Genètica Microbiologia i Estadística Facultat de Biologia Universitat de Barcelona Barcelona Spain

Department of Biochemistry and Molecular Biophysics Columbia University New York NY USA

Department of Biochemistry and Molecular Genetics University of Colorado Anschutz Medical Campus Aurora CO USA

Department of Biochemistry McGill University Montréal Quebec Canada

Department of Bioinformatics and Computational Biology Division of Quantitative Sciences the University of Texas MD Anderson Cancer Center Houston TX USA

Department of Biological Sciences Carnegie Mellon University Pittsburgh PA USA

Department of Biological Sciences University of Maryland Baltimore County Baltimore MD USA

Department of Biology Texas A and M University College Station TX USA

Department of Bionanoscience Delft University of Technology Delft the Netherlands

Department of Cell Biology and Molecular Genetics University of Maryland College Park MD USA

Department of Chemistry and Biochemistry University of Notre Dame Notre Dame IN USA

Department of Chemistry Biochemistry and Pharmaceutical Sciences University of Bern Bern Switzerland

Department of Computational Biology and Medical Sciences Graduate School of Frontier Sciences the University of Tokyo Kashiwa Japan

Department of Human Biology University of Haifa Haifa Israel

Department of Medicine Harvard Medical School Boston MA USA

Department of Molecular and Cellular Biology College of Biological Sciences University of California Davis Davis CA USA

Department of Molecular Biophysics and Biochemistry Yale School of Medicine New Haven CT USA

Department of Oncology Pathology Science for Life Laboratory Karolinska Institute Stockholm Sweden

Department of Pharmaceutical Sciences College of Pharmacy University of Illinois Chicago Chicago IL USA

Department of Physics Li Ka Shing Institute of Virology and Centre for Prions and Protein Folding Diseases University of Alberta Edmonton Alberta Canada

Division of Experimental Medicine McGill University Montréal Quebec Canada

Division of Rheumatology Inflammation and Immunity Brigham and Women's Hospital Boston MA USA

Division of Virology Department of Pathology Addenbrookes Hospital University of Cambridge Cambridge UK

EIRNA Bio Bioinnovation Hub Cork Ireland

Faculty of Biology and Preclinical Medicine University of Regensburg Regensburg Germany

Gerald Bronfman Department of Oncology McGill University Montréal Quebec Canada

Helmholtz Institute for RNA based Infection Research Helmholtz Centre for Infection Research Würzburg Germany

Institute for Bioscience and Biotechnology Research Rockville MD USA

Institute for Integrative Biology of the Cell CEA Université Paris Saclay CNRS Gif sur Yvette France

Institute of Biochemistry and Molecular Biology University of Hamburg Hamburg Germany

Institute of Biophysics National Research Council Unit Povo Italy

Institute of Genetics and Biotechnology Faculty of Biology University of Warsaw Warsaw Poland

Laboratory of Regulation of Gene Expression Institute of Microbiology of the Czech Academy of Sciences Prague Czech Republic

Lady Davis Institute McGill University Montréal Quebec Canada

MRC Toxicology Unit University of Cambridge Cambridge UK

New York Structural Biology Center New York NY USA

Oncode Institute Hubrecht Institute KNAW and University Medical Center Utrecht Utrecht the Netherlands

RNA Bioscience Initiative University of Colorado Anschutz Medical Campus Aurora CO USA

RNA Systems Biochemistry Laboratory RIKEN Cluster for Pioneering Research Wako Japan

School of Biochemistry and Cell Biology University College Cork Cork Ireland

Shemyakin Ovchinnikov Institute of Bioorganic Chemistry RAS Moscow Russia

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