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Structural and functional insights into 5′-ppp RNA pattern recognition by the innate immune receptor RIG-I

Nature Structural & Molecular Biology volume 17, pages 781–787 (2010)Cite this article

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Abstract

RIG-I is a cytosolic helicase that senses 5′-ppp RNA contained in negative-strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies established that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5′-ppp RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD bound to both blunt ends of a self-complementary 5′-ppp dsRNA 12-mer, with interactions involving 5′-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD sequesters the observable 5′-pp of the bound RNA, with a stacked phenylalanine capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5′-ppp dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2′-OCH3 RNA modifications on the interferon response.

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Figure 1: Sequence alignment of RIG-I family of pattern-recognition receptors and ITC studies of the binding of RIG-I CTD domain to RNA as a function of 5′-end phosphorylation and duplex/strand status.
Figure 2: Details of the crystal structure of the RIG-I CTD bound to blunt-end 5′-pp dsRNA 12-mer.
Figure 3: Role of 5′-phosphorylated ends in the crystal structure of the RIG-I CTD bound to blunt-end 5′-pp dsRNA 12-mer and comparison of the electrostatics of the binding surfaces of RIG-I, MDA5 and LGP2 CTDs for 5′-ppp dsRNA.
Figure 4: ITC and electrophoretic mobility shift studies of the binding of blunt-end 5′-ppp dsRNA 12-mer to wild-type and mutants of RIG-I CTD.
Figure 5: In vivo analysis of the impact of point mutants on the biological activity of RIG-I.
Figure 6: In vivo analysis of 5′-ppp RNA strand 2′-OCH3 substituent effects.

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Acknowledgements

The authors are grateful to the staff of X-29 beamline at Brookhaven National Laboratory for their assistance during synchrotron data collection, D. Shimanshu and Y. Tian for their assistance in synchrotron data collection, M. Aigner for chemical synthesis of 5′-ppp RNAs undertaken in Innsbruck and G. Wardle for MS analysis in the RU proteomics facility to quality control the 5′-ppp RNAs. D.J.P. was supported by funds from the Abby Rockefeller Mauze Trust and the Maloris Foundation. R.M. was supported by the Austrian Science fund FWF (I317). G.H. was supported by grants from the Bundesministerium für Bildung und Forschung Biofuture and GoBio and from the Deutsche Forschungsgemeinschaft (SFB704, SFB670, SFB832 and KFO177).

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Author notes

  1. Yanli Wang and Janos Ludwig: These authors contributed equally to this work.

Authors and Affiliations

  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Yanli Wang, Haitao Li, Gang Sheng & Dinshaw J Patel

  2. Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, University of Bonn, Bonn, Germany

    Janos Ludwig, Christine Schuberth, Marion Goldeck, Martin Schlee & Gunther Hartmann

  3. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York, USA

    Stefan Juranek & Thomas Tuschl

  4. Institute for Organic Chemistry, Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria

    Ronald Micura

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Contributions

Y.W. expressed the RIG-I CTD domain, with G.S. assisting with protein expression and purification; Y.W. crystallized and solved the structure of the complex; J.L. was responsible for chemical synthesis of palindromic and non-palindromic 5′-ppp RNAs and 2′-O-methyl-modified 5′-ppp RNAs; C.S., M.G. and M.S. were responsible for the in vivo functional assays, including expression of full-length RIG-I mutants; H.L. and Y.W. were responsible for ITC titration assays; S.J. was responsible for gel-shift assays and prepared the plasmids for mutant RIG-I CTD expression; R.M. provided earlier batches of short 5′-ppp RNAs for crystallization trials; the paper was written by D.J.P., G.H. and T.T. with the assistance of the other authors.

Corresponding authors

Correspondence to Thomas Tuschl, Gunther Hartmann or Dinshaw J Patel.

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Wang, Y., Ludwig, J., Schuberth, C. et al. Structural and functional insights into 5′-ppp RNA pattern recognition by the innate immune receptor RIG-I. Nat Struct Mol Biol 17, 781–787 (2010). https://doi.org/10.1038/nsmb.1863

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