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Guidelines for minimal reporting requirements, design and interpretation of experiments involving the use of eukaryotic dual gene expression reporters (MINDR)

Nature Structural & Molecular Biology volume 32, pages 418–430 (2025)Cite this article

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Abstract

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.

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Fig. 1: Principles of a dual-reporter strategy and the most common artifacts (shaded background) arising from unexamined assumptions.
Fig. 2: Assessment of dual-reporter transcriptional integrity.
Fig. 3: Fused versus unfused dual reporters.
Fig. 4: Challenges of dual-reporter assays in the study of IRES activity.

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Data availability

No original data have been generated or reanalyzed during the development of these guidelines.

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Acknowledgements

We acknowledge many colleagues who informally supported and encouraged the development of these guidelines. The following authors acknowledge funding support: P.V.B. by the Wellcome Trust (210692/Z/18/]) and Science Foundation Ireland (20/FFP-A/8929); L.S.V. by the Lead Agency (DFG and CSF) (grant 23-08669L) and the Praemium Academiae grant provided by the Czech Academy of Sciences and CZ.02.01.01/00/22_008/0004575 (RNA for therapy by ERDF and MEYS); S.E.D. from the Russian Science Foundation (23-14-00218, the IRES and cell-free sections); C.S.F. by the NIH (R35 GM152137); W.V.G. by the NIH (R01GM132358); Y.C. by the NIH (R01GM130838); J.F.A. by the Irish Research Council (IRCLA/2019/74); D.E.A. by the Russian Science Foundation (20-14-00121, RNA versus DNA transfection section); M. Mariotti by RYC2019-027746-I, PID2020-115122GA-I00 and PID2023-147164NB-I00 by MICIU/AEI (Ministry of Science, Innovation and Universities; State Research Agency of Spain) /10.13039/501100011033 and “ESF Investing in your future.; M.S.S. by the NIH (R01GM148702). J.K. by National Science Centre (UMO-2021/41/B/NZ2/03036). J.S.K. by NIH (R35GM118070). P.L.C. by the NIH (DP1GM146256). S.I. by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (JP24H02307). M. Mikl by ISF personal grant (2219/22).

Author information

Author notes

  1. Gary Loughran

    Present address: EIRNA Bio, Bioinnovation Hub, Cork, Ireland

Authors and Affiliations

  1. School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland

    Gary Loughran, Martina M. Yordanova, John F. Atkins & Pavel V. Baranov

  2. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

    Dmitry E. Andreev, Ilya M. Terenin, Ivan N. Shatsky & Sergey E. Dmitriev

  3. Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia

    Dmitry E. Andreev

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

    Olivier Namy

  5. Department of Human Biology, University of Haifa, Haifa, Israel

    Martin Mikl

  6. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA

    C. Joel McManus

  7. Division of Virology, Department of Pathology, Addenbrookes Hospital, University of Cambridge, Cambridge, UK

    Andrew E. Firth

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

    Christopher S. Fraser

  9. Institute of Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany

    Zoya Ignatova

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

    Shintaro Iwasaki

  11. RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Japan

    Shintaro Iwasaki

  12. Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland

    Joanna Kufel

  13. Department of Oncology–Pathology, Science for Life Laboratory, Karolinska Institute, Stockholm, Sweden

    Ola Larsson

  14. Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland

    Sebastian A. Leidel & Oliver Mühlemann

  15. Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois Chicago, Chicago, IL, USA

    Alexander S. Mankin & Nora Vázquez-Laslop

  16. Center for Biomolecular Sciences, University of Illinois Chicago, Chicago, IL, USA

    Alexander S. Mankin

  17. Departament de Genètica, Microbiologia i Estadística, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain

    Marco Mariotti

  18. Oncode Institute, Hubrecht Institute—KNAW and University Medical Center Utrecht, Utrecht, the Netherlands

    Marvin E. Tanenbaum

  19. Department of Bionanoscience, Delft University of Technology, Delft, the Netherlands

    Marvin E. Tanenbaum

  20. Lady Davis Institute, McGill University, Montréal, Quebec, Canada

    Ivan Topisirovic

  21. Gerald Bronfman Department of Oncology, McGill University, Montréal, Quebec, Canada

    Ivan Topisirovic

  22. Department of Biochemistry, McGill University, Montréal, Quebec, Canada

    Ivan Topisirovic & Nahum Sonenberg

  23. Division of Experimental Medicine, McGill University, Montréal, Quebec, Canada

    Ivan Topisirovic

  24. Institute of Biophysics, National Research Council (CNR) Unit, Povo, Italy

    Gabriela Viero

  25. Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection Research (HZI), Würzburg, Germany

    Neva Caliskan

  26. Faculty of Biology and Preclinical Medicine, University of Regensburg, Regensburg, Germany

    Neva Caliskan

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

    Yiwen Chen

  28. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA

    Patricia L. Clark

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

    Jonathan D. Dinman

  30. Institute for Bioscience & Biotechnology Research, Rockville, MD, USA

    Jonathan D. Dinman

  31. Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD, USA

    Philip J. Farabaugh

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

    Wendy V. Gilbert

  33. Division of Rheumatology, Inflammation and Immunity, Brigham and Women’s Hospital, Boston, MA, USA

    Pavel Ivanov

  34. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Pavel Ivanov

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

    Jeffrey S. Kieft

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

    Jeffrey S. Kieft

  37. New York Structural Biology Center, New York, NY, USA

    Jeffrey S. Kieft

  38. Department of Biology, Texas A&M University, College Station, TX, USA

    Matthew S. Sachs

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

    Anna-Lena Steckelberg

  40. MRC Toxicology Unit, University of Cambridge, Cambridge, UK

    Anne E. Willis

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

    Michael T. Woodside

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

    Leos Shivaya Valasek

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Correspondence to Gary Loughran, Leos Shivaya Valasek, Sergey E. Dmitriev or Pavel V. Baranov.

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Competing interests

G.L. and P.V.B. are cofounders and shareholders of EIRNA Bio Ltd. The remaining authors declare no competing interests.

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Loughran, G., Andreev, D.E., Terenin, I.M. et al. Guidelines for minimal reporting requirements, design and interpretation of experiments involving the use of eukaryotic dual gene expression reporters (MINDR). Nat Struct Mol Biol 32, 418–430 (2025). https://doi.org/10.1038/s41594-025-01492-x

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