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Programmable protein expression using a genetically encoded m6A sensor

Nature Biotechnology (2024)Cite this article

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Abstract

The N6-methyladenosine (m6A) modification is found in thousands of cellular mRNAs and is a critical regulator of gene expression and cellular physiology. m6A dysregulation contributes to several human diseases, and the m6A methyltransferase machinery has emerged as a promising therapeutic target. However, current methods for studying m6A require RNA isolation and do not provide a real-time readout of mRNA methylation in living cells. Here we present a genetically encoded m6A sensor (GEMS) technology, which couples a fluorescent signal with cellular mRNA methylation. GEMS detects changes in m6A caused by pharmacological inhibition of the m6A methyltransferase, giving it potential utility for drug discovery efforts. Additionally, GEMS can be programmed to achieve m6A-dependent delivery of custom protein payloads in cells. Thus, GEMS is a versatile platform for m6A sensing that provides both a simple readout for m6A methylation and a system for m6A-coupled protein expression.

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Fig. 1: Overview of the GEMS system.
Fig. 2: GEMS depends on m6A recognition.
Fig. 3: GEMS is METTL3 dependent.
Fig. 4: GEMS detects differences in methylation across cell types.
Fig. 5: GEMS senses changes in m6A caused by small-molecule inhibition of METTL3.
Fig. 6: m6A-coupled effector protein delivery counteracts the effects of m6A hypermethylation in cancer cells.

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All data supporting the findings of this study are available within the paper and its Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank members of the Meyer laboratory for helpful discussions and feedback. We are grateful to the Duke Cancer Institute Flow Cytometry Core and the Duke Functional Genomics Core for providing the infrastructure and support for cell sorting experiments. This work was supported by the Rita Allen Foundation and the National Institutes of Health (R01MH118366, DP1DA046584 and RM1HG011563) to K.D.M.

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Authors and Affiliations

  1. Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA

    Bahjat F. Marayati & Kate D. Meyer

  2. Department of Integrative Immunobiology, Duke University School of Medicine, Durham, NC, USA

    Matthew G. Thompson & Stacy M. Horner

  3. Department of Medicine, Duke University School of Medicine, Durham, NC, USA

    Christopher L. Holley & Stacy M. Horner

  4. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA

    Christopher L. Holley

  5. Department of Neurobiology, Duke University School of Medicine, Durham, NC, USA

    Kate D. Meyer

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Contributions

K.D.M. conceived of the study, designed the GEMS system and performed initial characterization. B.F.M. and K.D.M. planned experiments and further developed the system. B.F.M. conducted experiments and analyzed data, with help from K.D.M. M.G.T. generated METTL3 degron cells, with guidance from S.M.H. C.L.H. oversaw ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) experiments to quantify m6A. K.D.M. and B.F.M. wrote the manuscript.

Corresponding author

Correspondence to Kate D. Meyer.

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Competing interests

K.D.M. has filed a patent application for the GEMS technology through Duke University. The remaining authors declare no competing interests.

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Supplementary Figs. 1–8

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Supplementary Data 1

Flow cytometry gating strategy used for cell sorting

Supplementary Data 2

Source Data for western blots presented in Supplementary Figures

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Source Data Figs. 2–4

Source Data for western blots presented in Figs. 2–4

Source Data Figs. 5 and 6

Source Data for western blots presented in Figs. 5 and 6

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Marayati, B.F., Thompson, M.G., Holley, C.L. et al. Programmable protein expression using a genetically encoded m6A sensor. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-023-01978-3

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