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Molecular characterization of endosomal self RNA Rmrp-engaged TLR3 dimerization to prime innate activation

Cell Research volume 35, pages 824–839 (2025)Cite this article

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Abstract

The pre-dimerization of endosome-localized RNA sensor Toll-like receptor 3 (TLR3) is required for its innate recognition, yet how TLR3 pre-dimers are formed and precisely primed for innate activation remains unclear. Here, we demonstrate that endosome-localized self RNA Rmrp directly binds to TLR3 and induces TLR3 dimerization in the early endosome but does not interact with endosome-localized TLR7, TLR8, TLR9 or cytoplasmic RNA sensor RIG-I under homeostatic conditions. Cryo-EM structure of Rmrp–TLR3 complex reveals a novel lapped conformation of TLR3 dimer engaged by Rmrp, which is distinct from the activation mechanism by dsRNA and the specific structural feature at the 3’-end of Rmrp is critical for its functional interaction with TLR3. Furthermore, K42 residue of TLR3 is essential for binding to Rmrp and subsequent dimerization. Rmrp dissociates from TLR3 following endosomal acidification, generating a matured TLR3 dimer which is primed for innate recognition and activation. Myeloid-cell deficiency of Rmrp reduces TLR3 dimerization and attenuates TLR3-mediated antiviral responses against influenza A both in vitro and in vivo. These findings elucidate the structural mode of self RNA Rmrp-primed TLR3 dimerization and ready for efficient innate recognition on endosomal membrane, extending our knowledge of how membrane-associated TLRs pre-dimerize and suggesting a new function of subcellular localized self RNAs in empowering innate activation.

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Fig. 1: Endosomal RNA Rmrp directly binds to innate sensor TLR3.
Fig. 2: Rmrp specifically interacts with full-length TLR3 in early endosome.
Fig. 3: Rmrp is required for TLR3 dimerization.
Fig. 4: Rmrp enables TLR3 innate activation.
Fig. 5: Cryo-EM structure of Rmrp-C–mTLR3 complex.
Fig. 6: K42 residue of TLR3 is essential for binding to Rmrp.
Fig. 7: Rmrp promotes TLR3-initiated antiviral innate response in vitro and in vivo.

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Data availability

All data are available in the main text or supplementary materials. RNA-seq and iCLIP data are deposited in the Gene Expression Omnibus under accession number GSE159459. The 3D cryo-EM density maps have been deposited into the Electron Microscopy Data Bank under accession numbers EMD-30624, EMD-30626. The coordinates have been deposited into the Protein Data Bank under accession numbers 7DA7, 7DAS.

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Acknowledgements

We thank Drs. Quan Wang and Yan Gao for technical assistance. This work was supported by grants from the National Natural Science Foundation of China (82388201) and CAMS Innovation Fund for Medical Sciences (2024-I2M-ZD-005, 2021-I2M-1-017).

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  1. These authors contributed equally: Shikun Zhang, Bo Li, Lun Liu.

Authors and Affiliations

  1. Department of Immunology, Center for Immunotherapy, Institute of Basic Medical Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China

    Shikun Zhang, Lun Liu, Dongsheng Gong & Xuetao Cao

  2. State Key Laboratory of Medicinal Chemical Biology, Institute of Immunology, College of Life Sciences, Nankai University, Tianjin, China

    Bo Li, Deyu Zhang, Hua Qin, Deling Kong, Zihe Rao & Xuetao Cao

  3. Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Fengjiang Liu, Xiuna Yang & Zihe Rao

  4. National Infrastructures for Translational Medicine, Institute of Clinical Medicine, Peking Union Medical College Hospital, Beijing, China

    Shuyang Zhang

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Contributions

X.C. designed and supervised the study. Shikun Z., B.L., L.L., D.G., D.Z., F.L., X.Y., H.Q. performed the experiments. L.L. generated Rmrpfl/fl mice. D.K. and Shuyang Z. provided reagents and helpful discussion. Shikun Z., B.L., Z.R. and X.C. analyzed the data and wrote the manuscript.

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Correspondence to Zihe Rao or Xuetao Cao.

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Zhang, S., Li, B., Liu, L. et al. Molecular characterization of endosomal self RNA Rmrp-engaged TLR3 dimerization to prime innate activation. Cell Res 35, 824–839 (2025). https://doi.org/10.1038/s41422-025-01178-5

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