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Structural basis for duplex RNA recognition and cleavage by Archaeoglobus fulgidus C3PO

Nature Structural & Molecular Biology volume 20, pages 380–386 (2013)Cite this article

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Abstract

Oligomeric complexes of Trax and Translin proteins, known as C3POs, participate in several eukaryotic nucleic acid metabolism pathways, including RNA interference and tRNA processing. In RNA interference in humans and Drosophila, C3PO activates the RNA-induced silencing complex (RISC) by removing the passenger strand of the small interfering RNA precursor duplex, using nuclease activity present in Trax. How C3POs engage with nucleic acid substrates is unknown. Here we identify a single protein from Archaeoglobus fulgidus that assembles into an octamer highly similar to human C3PO. The structure in complex with duplex RNA reveals that the octamer entirely encapsulates a single 13-base-pair RNA duplex inside a large inner cavity. Trax-like-subunit catalytic sites target opposite strands of the duplex for cleavage separated by 7 base pairs. The structure provides insight into the mechanism of RNA recognition and cleavage by an archaeal C3PO-like complex.

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Figure 1: Overview of AfC3PO–duplex RNA structure.
Figure 2: AfC3PO Trax-like-subunit catalytic sites.
Figure 3: AfC3PO–duplex RNA interactions.
Figure 4: The four tetrameric inner surfaces of the AfC3PO cavity.
Figure 5: Conformational differences between AfC3PO Trax-like and Translin-like subunits.
Figure 6: AfC3PO duplex-RNA cleavage assays.

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Acknowledgements

We thank staff at Diamond Light Source, UK for help with data collection. This work was funded by a UK Medical Research Council Career Development Award to J.S.P. (grant no. G0600097). The pTwo-E vector was a gift from A. Oliver, University of Sussex, Brighton, UK.

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  1. Department of Biochemistry, University of Oxford, Oxford, UK

    Eneida A Parizotto, Edward D Lowe & James S Parker

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  1. Eneida A Parizotto
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Contributions

E.A.P. produced and purified the proteins, grew the crystals and performed the biochemical assays. E.D.L. provided advice, maintained facilities and assisted with X-ray data collection. J.S.P. collected X-ray data, determined the structures and wrote the manuscript.

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Correspondence to James S Parker.

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Parizotto, E., Lowe, E. & Parker, J. Structural basis for duplex RNA recognition and cleavage by Archaeoglobus fulgidus C3PO. Nat Struct Mol Biol 20, 380–386 (2013). https://doi.org/10.1038/nsmb.2487

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