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Structural insights into double-stranded RNA recognition and transport by SID-1

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

RNA uptake by cells is critical for RNA-mediated gene interference (RNAi) and RNA-based therapeutics. In Caenorhabditis elegans, RNAi is systemic as a result of SID-1-mediated double-stranded RNA (dsRNA) across cells. Despite the functional importance, the underlying mechanisms of dsRNA internalization by SID-1 remain elusive. Here we describe cryogenic electron microscopy structures of SID-1, SID-1–dsRNA complex and human SID-1 homologs SIDT1 and SIDT2, elucidating the structural basis of dsRNA recognition and import by SID-1. The homodimeric SID-1 homologs share conserved architecture, but only SID-1 possesses the molecular determinants within its extracellular domains for distinguishing dsRNA from single-stranded RNA and DNA. We show that the removal of the long intracellular loop between transmembrane helix 1 and 2 attenuates dsRNA uptake and systemic RNAi in vivo, suggesting a possible endocytic mechanism of SID-1-mediated dsRNA internalization. Our study provides mechanistic insights into dsRNA internalization by SID-1, which may facilitate the development of dsRNA applications based on SID-1.

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Fig. 1: Function and architecture of SID-1 homologs.
Fig. 2: Assembly interface of SID-1.
Fig. 3: EM structure of SID-1–dsRNA complex.
Fig. 4: Structural basis of dsRNA recognition by SID-1.
Fig. 5: Potential mechanism of SID-1-mediated dsRNA uptake.

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Data availability

The UniProt accession codes for the sequences of C. elegans sid-1, Homo sapiens sidt1 and sidt2 are Q9GZC8, Q9NXL6 and Q8NBJ9, respectively. The coordinates of 50 bp dsRNA fragment were downloaded from the Protein Data Bank (PDB 7W0E). The three-dimensional cryo-EM density maps of the SID-1, SID-1–dsRNA, hSIDT1 and hSIDT2 have been deposited in the Electron Microscopy Data Bank under accession codes EMD-34825, EMD-34850, EMD-36008 and EMD-36009, respectively. The coordinates of the SID-1 and SID-1–dsRNA, hSIDT1 and hSIDT2 have been deposited in the Protein Data Bank under accession codes PDB 8HIP, PDB 8HKE, PDB 8J6M and PDB 8J6O, respectively. Source data are provided with this paper.

References

  1. Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Hannon, G. J. RNA interference. Nature 418, 244–251 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Agrawal, N. et al. RNA interference: biology, mechanism, and applications. Microbiol. Mol. Biol. Rev. 67, 657–685 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kim, D. & Rossi, J. RNAi mechanisms and applications. Biotechniques 44, 613–616 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. Mello, C. C. & Conte, D. Revealing the world of RNA interference. Nature 431, 338–342 (2004).

    Article  CAS  PubMed  Google Scholar 

  7. Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Macrae, I. J. et al. Structural basis for double-stranded RNA processing by Dicer. Science 311, 195–198 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001).

    Article  CAS  PubMed  Google Scholar 

  10. Hutvagner, G. et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838 (2001).

    Article  CAS  PubMed  Google Scholar 

  11. Elbashir, S. M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. & Hannon, G. J. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Pillai, R. S. et al. Inhibition of translational initiation by Let-7 microRNA in human cells. Science 309, 1573–1576 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Tabara, H., Grishok, A. & Mello, C. C. RNAi in C. elegans: soaking in the genome sequence. Science 282, 430–431 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. Timmons, L. & Fire, A. Specific interference by ingested dsRNA. Nature 395, 854 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Winston, W. M., Molodowitch, C. & Hunter, C. P. Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science 295, 2456–2459 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Winston, W. M., Sutherlin, M., Wright, A. J., Feinberg, E. H. & Hunter, C. P. Caenorhabditis elegans SID-2 is required for environmental RNA interference. Proc. Natl Acad. Sci. USA 104, 10565–10570 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Timmons, L., Tabara, H., Mello, C. C. & Fire, A. Z. Inducible systemic RNA silencing in Caenorhabditis elegans. Mol. Biol. Cell. 14, 2972–2983 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tijsterman, M., May, R. C., Simmer, F., Okihara, K. L. & Plasterk, R. H. Genes required for systemic RNA interference in Caenorhabditis elegans. Curr. Biol. 14, 111–116 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Saleh, M. C. et al. The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing. Nat. Cell Biol. 8, 793–802 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hinas, A., Wright, A. J. & Hunter, C. P. SID-5 is an endosome-associated protein required for efficient systemic RNAi in C. elegans. Curr. Biol. 22, 1938–1943 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bhatia, S. & Hunter, C. P. SID-4/NCK-1 is important for dsRNA import in Caenorhabditis elegans. G3 12, jkac252 (2022).

  23. Feinberg, E. H. & Hunter, C. P. Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301, 1545–1547 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Song, Y. et al. The functions of SID1 transmembrane family, member 2 (Sidt2). FEBS J. 290, 4626–4637 (2023).

    Article  CAS  PubMed  Google Scholar 

  25. Andaleon, A., Mogil, L. S. & Wheeler, H. E. Gene-based association study for lipid traits in diverse cohorts implicates BACE1 and SIDT2 regulation in triglyceride levels. PeerJ 6, e4314 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Chang, G. et al. SIDT2 is involved in the NAADP-mediated release of calcium from insulin secretory granules. J. Mol. Endocrinol. 56, 249–259 (2016).

    Article  CAS  PubMed  Google Scholar 

  27. Gombojav, B. et al. Multiple susceptibility loci at chromosome 11q23.3 are associated with plasma triglyceride in East Asians. J. Lipid Res. 57, 318–324 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. León-Reyes, G. et al. The variant rs1784042 of the SIDT2 gene is associated with metabolic syndrome through low HDL-c levels in a Mexican population. Genes 11, 1192 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Moon, S., Lee, Y., Won, S. & Lee, J. Multiple genotype–phenotype association study reveals intronic variant pair on SIDT2 associated with metabolic syndrome in a Korean population. Hum. Genomics 12, 48 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chen, Q. et al. SIDT1-dependent absorption in the stomach mediates host uptake of dietary and orally administered microRNAs. Cell Res. 31, 247–258 (2021).

    Article  PubMed  Google Scholar 

  31. Aizawa, S. et al. Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes. Autophagy 13, 218–222 (2017).

    Article  CAS  PubMed  Google Scholar 

  32. Nguyen, T. A. et al. SIDT2 transports extracellular dsRNA into the cytoplasm for innate immune recognition. Immunity 47, 498–509.e6 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mendez-Acevedo, K. M., Valdes, V. J., Asanov, A. & Vaca, L. A novel family of mammalian transmembrane proteins involved in cholesterol transport. Sci. Rep. 7, 7450 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Whangbo, J. S., Weisman, A. S., Chae, J. & Hunter, C. P. SID-1 domains important for dsRNA import in Caenorhabditis elegans. G3 7, 3887–3899 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Shih, J. D., Fitzgerald, M. C., Sutherlin, M. & Hunter, C. P. The SID-1 double-stranded RNA transporter is not selective for dsRNA length. RNA 15, 384–390 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Pratt, A. J., Rambo, R. P., Lau, P. W. & MacRae, I. J. Preparation and characterization of the extracellular domain of human Sid-1. PLoS ONE 7, e33607 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Li, W., Koutmou, K. S., Leahy, D. J. & Li, M. Systemic RNA interference deficiency-1 (SID-1) extracellular domain selectively binds long double-stranded RNA and is required for RNA transport by SID-1. J. Biol. Chem. 290, 18904–18913 (2015).

  38. Shih, J. D. & Hunter, C. P. SID-1 is a dsRNA-selective dsRNA-gated channel. RNA 17, 1057–1065 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. McEwan, D. L., Weisman, A. S. & Hunter, C. P. Uptake of extracellular double-stranded RNA by SID-2. Mol. Cell 47, 746–754 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Whangbo, J. S. & Hunter, C. P. Environmental RNA interference. Trends Genet. 24, 297–305 (2008).

    Article  CAS  PubMed  Google Scholar 

  41. Nguyen, T. A. et al. SIDT1 localizes to endolysosomes and mediates double-stranded RNA transport into the cytoplasm. J. Immunol. 202, 3483–3492 (2019).

    Article  CAS  PubMed  Google Scholar 

  42. Itoh, K., Watanabe, A., Funami, K., Seya, T. & Matsumoto, M. The clathrin-mediated endocytic pathway participates in dsRNA-induced IFN-β production. J. Immunol. 181, 5522–5529 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Jialin, G., Xuefan, G. & Huiwen, Z. SID1 transmembrane family, member 2 (Sidt2): a novel lysosomal membrane protein. Biochem. Biophys. Res. Commun. 402, 588–594 (2010).

    Article  PubMed  Google Scholar 

  44. Wienken, C. J., Baaske, P., Rothbauer, U., Braun, D. & Duhr, S. Protein-binding assays in biological liquids using microscale thermophoresis. Nat. Commun. 1, 100 (2010).

    Article  PubMed  Google Scholar 

  45. Aizawa, S. et al. Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes. Autophagy 12, 565–578 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Auld, D. S. Zinc coordination sphere in biochemical zinc sites. Biometals 14, 271–313 (2001).

    Article  CAS  PubMed  Google Scholar 

  47. Vasiliauskaité-Brooks, I. et al. Structural insights into adiponectin receptors suggest ceramidase activity. Nature 544, 120–123 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Pei, J., Millay, D. P., Olson, E. N. & Grishin, N. V. CREST—a large and diverse superfamily of putative transmembrane hydrolases. Biol. Direct 6, 37 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Fierro-Monti, I. & Mathews, M. B. Proteins binding to duplexed RNA: one motif, multiple functions. Trends Biochem. Sci. 25, 241–246 (2000).

    Article  CAS  PubMed  Google Scholar 

  50. Saunders, L. R. & Barber, G. N. The dsRNA binding protein family: critical roles, diverse cellular functions. FASEB J. 17, 961–983 (2003).

    Article  CAS  PubMed  Google Scholar 

  51. Masliah, G., Barraud, P. & Allain, F. H. RNA recognition by double-stranded RNA binding domains: a matter of shape and sequence. Cell. Mol. Life Sci. 70, 1875–1895 (2013).

    CAS  PubMed  Google Scholar 

  52. Vicens, Q. & Kieft, J. S. Thoughts on how to think (and talk) about RNA structure. Proc. Natl Acad. Sci. USA 119, e2112677119 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Bevilacqua, P. C. & Cech, T. R. Minor-groove recognition of double-stranded RNA by the double-stranded RNA-binding domain from the RNA-activated protein kinase PKR. Biochemistry 35, 9983–9994 (1996).

    Article  CAS  PubMed  Google Scholar 

  54. Pérez, A., Noy, A., Lankas, F., Luque, F. J. & Orozco, M. The relative flexibility of B-DNA and A-RNA duplexes: database analysis. Nucleic Acids Res. 32, 6144–6151 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Contu, V. R. et al. Lysosomal targeting of SIDT2 via multiple Yxxϕ motifs is required for SIDT2 function in the process of RNautophagy. J. Cell Sci. 130, 2843–2853 (2017).

    CAS  PubMed  Google Scholar 

  56. Vuković, L., Koh, H. R., Myong, S. & Schulten, K. Substrate recognition and specificity of double-stranded RNA binding proteins. Biochemistry 53, 3457–3466 (2014).

    Article  PubMed  Google Scholar 

  57. Qian, D. et al. Structural insight into the human SID1 transmembrane family member 2 reveals its lipid hydrolytic activity. Nat. Commun. 14, 3568 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Mogurampelly, S. & Maiti, P. K. Translocation and encapsulation of siRNA inside carbon nanotubes. J. Chem. Phys. 138, 034901 (2013).

    Article  PubMed  Google Scholar 

  59. Perera, R. T. et al. Unzipping of A-form DNA-RNA, A-form DNA-PNA, and B-form DNA-DNA in the α-hemolysin nanopore. Biophys. J. 110, 306–314 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Huang, X. et al. The landscape of mRNA nanomedicine. Nat. Med. 28, 2273–2287 (2022).

    Article  CAS  PubMed  Google Scholar 

  61. Rohner, E., Yang, R., Foo, K. S., Goedel, A. & Chien, K. R. Unlocking the promise of mRNA therapeutics. Nat. Biotechnol. 40, 1586–1600 (2022).

    Article  CAS  PubMed  Google Scholar 

  62. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Zivanov, J. et al. New tools for automated high-resolution cryo-EM determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  66. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  67. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  68. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank X. Huang, B. Zhu, X. Li, L. Chen and other staff members at the Center for Biological Imaging (CBI), Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Science (IBP, CAS), for the support in cryo-EM data collection. We thank N. Zheng at the University of Washington for his helpful discussion and W. Fan for her research assistance. This work is funded by the National Natural Science Foundation of China (T2221001 to Y.L., M. Li and D.J.; 32271272 to D.J.; 12090051 to M. Li; and 82071851 to J.G.), the Institute of Physics, Chinese Academy of Sciences (E0VK101 and E2V4101 to D.J.), and the program for HUST Academic Frontier Youth Team (5001170068 to J.G.).

Author information

Author notes

  1. These authors contributed equally: Jiangtao Zhang, Chunhua Zhan, Junping Fan, Dian Wu, Ruixue Zhang.

Authors and Affiliations

  1. College of Life Science and Technology, Key Laboratory of Molecular Biophysics of MOE, Huazhong University of Science and Technology, Wuhan, Hubei, China

    Jiangtao Zhang, Chunhua Zhan, Dian Wu & Jianke Gong

  2. Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China

    Jiangtao Zhang, Di Wu, Xinyao Chen, Ying Lu, Ming Li & Daohua Jiang

  3. State Key Laboratory of Natural and Biomimetic Drugs, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing, China

    Junping Fan

  4. Key Laboratory of Agricultural Microbiome (MARA), Biotechnology Research Institute, Chinese Academy Agricultural Sciences, Beijing, China

    Ruixue Zhang

  5. University of Chinese Academy of Sciences, Beijing, China

    Di Wu, Xinyao Chen, Ying Lu, Ming Li & Daohua Jiang

  6. Food Laboratory of Zhongyuan, College of Agriculture, Henan University, Kaifeng, Henan, China

    Min Lin

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Contributions

D.J. and J.G. designed the experiments. J.Z. prepared the sample for the cryo-EM study and made all the constructs. J.Z. and J.F. collected cryo-EM data. J.F. and J.Z. processed the data and built and refined the models. Dian Wu performed the luciferase activity assay in S2 cells. C.Z. performed the in vivo RNAi assay. R.Z. and J.Z. performed the MST binding assay. J.Z. and X.C. performed the confocal imaging. J.Z., Di Wu and C.Z. prepared figures. Y.L., M. Li, M. Lin, J.G. and D.J. analyzed and interpreted the results. D.J. wrote the paper and all authors reviewed and revised the paper.

Corresponding authors

Correspondence to Jianke Gong or Daohua Jiang.

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Nature Structural & Molecular Biology thanks Ian Macrae, Gary Ruvkun and Hong-Wei Wang for their contribution to the peer review of this work. Primary Handling Editor: Sara Osman, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Expression and purification of SID-1 variants.

a-c, Expression levels of SID-1 variants in insect S2 cells by western blot. The S2 cells were prepared under the same conditions to the cells used for luciferase activity assay. n = 3 independent experiments. d-f, The representative gel filtration profiles of purified SID-1, hSIDT1, and SIDT2 for cryo-EM anaylsis. n = 3 independent experiments. g-k, Purificaiton of SID-13A (g), SID-15E (h), SID-1D70A/D171A (i), SID-1R172W/A173T (j), and SID-1L353-S423 (k) for dsRNA MST binding assay. n = 3 independent experiments. l, Cellular localization of SID-1 and SID-1ΔL353-S423 by confocal imaging. Cell membrane was stained by Dil (red). A GFP was fused to SID-1 and SID-1ΔL353-S423 for imaging, respectively. n = 3 independent experiments. Scale bars, 10 μm.

Source data

Extended Data Fig. 2 Cryo-EM data processing of SID-1 and SID-1/dsRNA complex.

a and e, Flowchart of cryo-EM data processing for SID-1 apo (a) and SID-1/dsRNA complex (e). Particle picking, 2D classification, and 3D classification were performed to remove bad particles, followed by 3D AutoRefine and Bayesian polish to improve the map quality. The polished particles were imported into CryoSPARC for further 3D classification and refinement. The final EM density maps were generated by the non-uniform (NU) refinement in CryoSPARC. b and f, Local resolution distribution of the final map of the SID-1 apo (b) and SID-1/dsRNA complex (f). c and g, Angular distribution of cryo-EM reconstruction of the SID-1 apo (c) and SID-1/dsRNA complex (g) used for the final refinements. d and h, Fourier shell correlations (FSC) curves of the SID-1 apo (d) and SID-1/dsRNA complex (h). The FSC curves of the final EM map without and with a solvent mask are colored in blue and red, respectively. Red arrow indicates the resolution of the map when FSC = 0.143. The FSC curve of the refined model fitting in the final EM map is colored in black. Black arrow indicates the resolution of model-map fitting when FSC = 0.5.

Extended Data Fig. 3 Cryo-EM data processing of human SIDT1 and SIDT2.

a and e, Flowchart of cryo-EM data processing for human SIDT1 (a) and SIDT2 (e). Particle picking, 2D classification, and 3D classification were performed to remove bad particles, followed by 3D AutoRefine and Bayesian polish to improve the map quality. The polished particles were imported into CryoSPARC for further 3D classification and refinement. The final EM maps were generated by the non-uniform (NU) refinement in CryoSPARC. b and f, Local resolution distribution of the final map of the human SIDT1 (b) and SIDT2 (f). c and g, Angular distribution of cryo-EM reconstruction of the human SIDT1 (c) and SIDT2 (g) used for the final refinements. d and h, Fourier shell correlations (FSC) curves of the human SIDT1 (d) and SIDT2 (h). The FSC curves of the final EM map without and with a solvent mask are colored in blue and red, respectively. Red arrow indicates the resolution of the map when FSC = 0.143. The FSC curve of the refined model fitting in the final EM map is colored in black. Black arrow indicates the resolution of model-map fitting when FSC = 0.5.

Extended Data Fig. 4 Representative EM densities of SID-1.

a to c, EM densities of SID-1 ECDI (a), ECDII (b), and TM helices (c) were shown as gray surface. Most residues shown side−chains in sticks.

Extended Data Fig. 5 Structural features of SID-1.

a, Topology of SID-1. SID-1 is colored from blue to red. Secondary structural elements are labelled. b, Structure of SID-1 monomer. c, Superposition of ECDI and ECDII. d, A cut-open surface representation of SID-1 TMD. e and f, Two short α-helices mediate the connection between ECDII and TMD. ECDI, ECDII, and TMD are colored in blue, pink, and green, respectively. Key residues on α5 and α6 shown side-chains in sticks. Red dashed lines indicate polar interactions.

Extended Data Fig. 6 Structural comparisons of SID-1 and human SIDT1 and SIDT2.

a and d, Superposition of SID-1 with hSIDT1 (a) and hSIDT2 (d). SID1, hSIDT1, and hSIDT2 are colored in green, pink, and brown, respectively. b and e, Superposition of ECD of SID-1 with ECD of hSIDT1 (b) and ECD of hSIDT2 (e). (c and f) Superposition of TMD of SID-1 with TMD of hSIDT1 (c) and TMD of hSIDT2 (f). g to i, Putative Zn2+ binding site in SID-1 (g), hSIDT1 (h), and hSIDT2 (i). Residues for ion coordinating shown side-chain in sticks. Yellow ball represents a putative Zn2+. EM densities for Zn2+ and key residues are shown in white surface.

Extended Data Fig. 7 Sequence alignment of SID-1 homologs.

Sequence alignment of C. elegans SID-1 and CHUP1, and human SIDT1 and SIDT2. Conserved residues were marked in blue. The putative Zn2+ binding site, dsRNA binding region I, region II, region III and the ion binding site in region II are highlighted in green, orange, red, yellow and cyan, respectively. The software Jalviewl.8.3 was used for sequence alignment.

Extended Data Fig. 8 Structural determinants for dsRNA binding.

a to c, Representative cryo-EM micrographs and 2D averages of SID1 (a), hSIDT1 (b), and hSIDT2 with or without dsRNA added to the sample. Yellow arrows indicate dsRNA. Scale bars represent 50 nm in EM micrographs and 10 nm in 2D averages, respectively. d, Key residues responsible for dsRNA binding in the ECD of SID-1. e and f, Residues at equilibrium positions of ECD of SID-1 (panel d) are highlighted in the ECD of hSIDT1 (e) and hSIDT2 (f). g, Superposition of SID-1apo and dsRNA bound SID-1. SID-1apo is colored in gray, the ECDI, ECDII, and TMD of dsRNA-bound SID-1 are colored in blue, pink, and green, respectively. (h to j) A closer look at the conformational changes in ECDI (h), ECDI and ECDII linker (i), and ECDII (j) between SID-1apo and dsRNA-bound protomer of SID-1dsRNA. Red arrows indicate local conformational shifts. (k to m) A closer look at the conformational changes in ECDI (k), ECDI and ECDII linker (l), and ECDII (m) between SID-1apo and dsRNA-unbound protomer of SID-1dsRNA.

Extended Data Fig. 9 Colocalization of SID-1 and dsRNA in C. elegans.

a, Colocalization of Cy5-labeled dsRNA and YFP::SID-1 in the body-wall muscle cells. The transgenic sid-1 (qt9) worms expressing YFP::SID-1 in the body-wall muscles and SID-1 in the pharynx muscles were imaged after the injection of Cy5-labeled dsRNA into the pharynx of the worms. b, Cy5-labeled dsRNA was not detected in the body-wall muscle cells of sid-1 (qt9) worms only expressing YFP::SID-1 in the body-wall muscle. c and d, No colocalization of SID-1 and cy5 (c) or cy5-ssRNA (d) was observed. Scale bars, 100 μm (upper panels), 10 μm (lower panels). e, Fraction of colocalization of cy5-dsRNA and YFP::SID-1 in observed sid-1 (qt9) worms. The number of independent repetitions in a-d is marked in the bar chart in panel e.

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Zhang, J., Zhan, C., Fan, J. et al. Structural insights into double-stranded RNA recognition and transport by SID-1. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01276-9

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