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Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain

Abstract

Short RNAs mediate gene silencing, a process associated with virus resistance, developmental control and heterochromatin formation in eukaryotes1,2,3,4,5. RNA silencing is initiated through Dicer-mediated processing of double-stranded RNA into small interfering RNA (siRNA)6,7. The siRNA guide strand associates with the Argonaute protein in silencing effector complexes, recognizes complementary sequences and targets them for silencing8,9,10,11. The PAZ domain is an RNA-binding module found in Argonaute and some Dicer proteins and its structure has been determined in the free state12,13,14. Here, we report the 2.6 Å crystal structure of the PAZ domain from human Argonaute eIF2c1 bound to both ends of a 9-mer siRNA-like duplex. In a sequence-independent manner, PAZ anchors the 2-nucleotide 3′ overhang of the siRNA-like duplex within a highly conserved binding pocket, and secures the duplex by binding the 7-nucleotide phosphodiester backbone of the overhang-containing strand and capping the 5′-terminal residue of the complementary strand. On the basis of the structure and on binding assays, we propose that PAZ might serve as an siRNA-end-binding module for siRNA transfer in the RNA silencing pathway, and as an anchoring site for the 3′ end of guide RNA within silencing effector complexes.

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Figure 1: Overview of the PAZ–siRNA-like duplex structure.
Figure 2: The PAZ–siRNA-like duplex subcomplex.
Figure 3: The details of PAZ–siRNA-like duplex interaction.
Figure 4: RNA-binding assays monitored using surface plasmon resonance.

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Acknowledgements

We thank K. Saigo for providing us with the eIF2C1 complementary DNA clone. This research was supported by the NIH. We thank Y. Cheng and personnel at the Advanced Photon Source (APS) beamlines 19BM and 14IDB for help in collecting the X-ray diffraction data. Use of the APS beamline was supported by the US Department of Energy, Basic Energy Sciences, Office of Science.

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Author notes

  1. Coordinates for the PAZ–siRNA complexes containing 2-nt ribo- and deoxyribonucleotide 3′ overhangs have been deposited in the Protein Data Bank under accession codes 1SI3 and 1SI2, respectively.

    • Keqiong Ye
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Correspondence to Dinshaw J. Patel.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Table 1

Crystallographic statistics (DOC 30 kb)

Supplementary Figure 1

Elution profiles of PAZ and PAZ-RNA complex. (JPG 26 kb)

Supplementary Figure 2

Stereo view of structural alignment of PAZ domains (JPG 62 kb)

Supplementary Figure 3

Sequence alignment of PAZ domains (JPG 172 kb)

Supplementary Figure Legends (DOC 26 kb)

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Ma, JB., Ye, K. & Patel, D. Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429, 318–322 (2004). https://doi.org/10.1038/nature02519

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  • DOI: https://doi.org/10.1038/nature02519

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