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Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

Nature volume 434, pages 666–670 (2005)Cite this article

Abstract

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism1,2,3 mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold4,5 critical for the endonuclease cleavage activity of RISC4,5,6. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5′-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5′ end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5′ end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5′ phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi–RNA complex, and that determined elsewhere7, provide direct support for the 5′ region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5′ end of the guide RNA.

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Figure 1: Crystal structure of the A. fulgidus Piwi–RNA complex.
Figure 2: The 5′-phosphate-binding site in the A. fulgidus Piwi–RNA complex.
Figure 3: Mutation studies of conserved 5′-phosphate-binding residues in A. fulgidus Piwi protein and human Ago2 protein.
Figure 4: Model of A. fulgidus Piwi protein (AfPiwi) bound to an 18-base-pair RNA duplex with 5′-phosphorylated single-nucleotide overhangs.

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Acknowledgements

We thank A. Saxena and personnel at synchrotron beamline X26C of the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory, for their assistance. Use of the NSLS beamline is supported by the US Department of Energy, Basic Energy Sciences, Office of Science. D.J.P. is supported by funds from the Abby Rockefeller Mauze Trust and the Dewitt Wallace and Maloris Foundations, and T.T. is supported by a National Institutes of Health grant.

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Authors and Affiliations

  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, 10021, USA

    Jin-Biao Ma, Yu-Ren Yuan & Dinshaw J. Patel

  2. Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York, 10021, USA

    Gunter Meister, Yi Pei & Thomas Tuschl

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  1. Jin-Biao Ma
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Correspondence to Dinshaw J. Patel.

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Supplementary Notes

Contains details of Supplementary Methods used, Supplementary Figures S1 and S2 and accompanying legends. It also contains Supplementary Table S1 and additional references. (PDF 548 kb)

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Ma, JB., Yuan, YR., Meister, G. et al. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434, 666–670 (2005). https://doi.org/10.1038/nature03514

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