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

Shikun Zhang^1,† , Bo Li^2,† , Lun Liu^1,† , Dongsheng Gong¹ , Deyu Zhang² , Fengjiang Liu³ , Xiuna Yang³ , Hua Qin² , Deling Kong² , Shuyang Zhang⁴ , Zihe Rao^2,3,* , Xuetao Cao^1,2

¹Department of Immunology, Center for Immunotherapy, Institute of Basic Medical Sciences, Peking U nion Medical College, Chinese Academy of Medical Sciences, Beijing, China
²State Key Laboratory of Medicinal Chemical Biology, Institute of Immunology, College of Life Sciences, Nankai University, Tianjin, China
³Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China
⁴National Infrastructures for Translational Medicine, Institute of Clinical Medicine, Peking U nion Medical College Hospital, Beijing, China
^†These authors contributed equally: Shikun Zhang, Bo Li, Lun Liu
* Correspondence: Zihe Rao(raozh@mail.tsinghua.edu.cn)

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.

https://doi.org/10.1038/s41422-025-01178-5

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