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A blue light receptor that mediates RNA binding and translational regulation

Abstract

Sensory photoreceptor proteins underpin light-dependent adaptations in nature and enable the optogenetic control of organismal behavior and physiology. We identified the bacterial light-oxygen-voltage (LOV) photoreceptor PAL that sequence-specifically binds short RNA stem loops with around 20 nM affinity in blue light and weaker than 1 µM in darkness. A crystal structure rationalizes the unusual receptor architecture of PAL with C-terminal LOV photosensor and N-terminal effector units. The light-activated PAL–RNA interaction can be harnessed to regulate gene expression at the RNA level as a function of light in both bacteria and mammalian cells. The present results elucidate a new signal-transduction paradigm in LOV receptors and conjoin RNA biology with optogenetic regulation, thereby paving the way toward hitherto inaccessible optoribogenetic modalities.

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Fig. 1: Photoactivated RNA-binding by the LOV receptor PAL (Uniprot C8XJT7).
Fig. 2: Structure of PAL in its dark-adapted state.
Fig. 3: Functional analysis of PAL.
Fig. 4: Light-dependent regulation of translation in mammalian cells.

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Data availability

Coordinates and structure-factor amplitudes of the PAL crystal structure that support the findings of this study have been deposited in the Protein Data Bank with the accession code 6HMJ. Additional raw data or materials are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank members of the Mayer and Möglich laboratories for discussion; H. Ruwe and C. Schmitz-Linneweber (HU Berlin) for help in the initial analysis of nucleic-acid binding; M. Humenik for assistance with SEC–MALS. Funding through a Sofja-Kovalevskaya Award of the Alexander-von-Humboldt Foundation (to A.M.); a Consolidator grant no. 615381 by the European Research Council (to G.M.) and Deutsche Forschungsgemeinschaft (nos. MO2192/6-1, MA3442/5-1, SFB-1089/P1 and Sfb-1078/B4) is gratefully acknowledged.

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Authors and Affiliations

Authors

Contributions

A.M.W. established the light-dependent SELEX protocol, conducted SELEX cycles 10–15, characterized PAL–RNA interactions via filter retention, Cherenkov measurements, RiboGreen fluorescence and surface plasmon resonance, designed eukaryotic reporter gene constructs, contributed to the sequence analysis of enrichment, established and performed switching experiments of PAL in eukaryotes and wrote the manuscript. J.K. purified PAL, characterized PAL–RNA interaction by fluorescence anisotropy, developed the bacterial reporter assay, conducted structure-function analysis, contributed to EPR experiments and wrote the manuscript. T.Z. isolated and cloned the PAL gene from N. multipartita; purified, biochemically analyzed and crystallized PAL and contributed to structure solution and refinement. She demonstrated preferential RNA binding by EMSA, adapted the light-dependent SELEX protocol, conducted SELEX cycles 1–9 and characterized PAL–RNA interactions via EMSA and RiboGreen fluorescence. S.P. designed, performed and analyzed reporter gene constructs for eukaryotic gene expression and RiboGreen assays of ANTAR sequences. C.R. designed, performed and analyzed reporter gene constructs for eukaryotic gene expression, performed sequence analysis of enrichment, established and performed switching experiments of PAL in eukaryotes. L.S. purified PAL and conducted fluorescence anisotropy experiments. G.P. designed, performed and analyzed reporter gene constructs for eukaryotic gene expression and contributed to Cherenkov measurements. S.M. solved and refined the PAL crystal structure. A.K. performed and analyzed reporter gene constructs for eukaryotic gene expression. M.J. purified PAL C284A and conducted EPR measurements. S.S. conducted EPR measurements and analyzed EPR data. L.L.B. performed sequence analysis of enrichment. C.S. advised on crystallography. R.B. supervised EPR measurements and analyzed EPR data. G.M. conceived, designed and supervised research and wrote the manuscript. A.M. identified the PAL gene, conceived, designed and supervised research and wrote the manuscript.

Corresponding authors

Correspondence to Günter Mayer or Andreas Möglich.

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Supplementary information

Supplementary Information

Supplementary Tables 1–4, Supplementary Figures 1–18

Reporting Summary

Supplementary Dataset 1

This dataset shows the progression and enrichment of individual RNA sequences through the SELEX selection cycles 3–15, as determined by RNA sequencing.

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Weber, A.M., Kaiser, J., Ziegler, T. et al. A blue light receptor that mediates RNA binding and translational regulation. Nat Chem Biol 15, 1085–1092 (2019). https://doi.org/10.1038/s41589-019-0346-y

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