Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes

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Abstract

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5′-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg2+-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide–target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3′-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.

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Figure 1: Crystal structure of T. thermophilus Ago(Asn 546) catalytic mutant bound to 5′-phosphorylated 21-nucleotide guide DNA and 12-nucleotide target RNA.
Figure 2: Crystal structure of T. thermophilus Ago(Glu 546) catalytic mutant bound to 5′-phosphorylated 21-nucleotide guide DNA and 15-nucleotide target RNA.
Figure 3: Crystal structure of T. thermophilus Ago(Asn 478) catalytic mutant bound to 5′-phosphorylated 21-nucleotide guide DNA and 19-nucleotide target RNA and identification of Mg 2+ binding sites within the catalytic pocket of the wild-type Ago complex.
Figure 4: Effect of complementarity and length on target DNA cleavage by T. thermophilus Ago.
Figure 5: Effect of sugar-phosphate backbone modifications on target DNA cleavage by T. thermophilus Ago.

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Protein Data Bank

Data deposits

The structures of ternary complexes of T. thermophilus Ago have been submitted to the Protein Data Bank. The accession codes are: 3HO1 (mutant Ago(Asn 546)–12-nucleotide target RNA), 3HJF (mutant Ago(Glu 546)–15-nucleotide target RNA), 3HK2 (mutant Ago(Asn 478)–19-nucleotide target RNA), 3HM9 (wild-type Ago–19-nucleotide target RNA, 50mMMg), 3HVR (wild-type Ago–19-nucleotide target RNA, 80mM Mg), and 3HXM (second crystal form of wild-type Ago–20-nucleotide target RNA containing twomismatches31).

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Acknowledgements

The research was supported by funds from the National Institutes of Health (NIH) and the Starr Foundation to D.J.P., and from the NIH, Starr Foundation and the Howard Hughes Medical Institute (HHMI) to T.T. We would like to thank the staff of NE-CAT beam lines at the Advanced Photon Source (APS), Argonne National Laboratory, and the X-29 beamline at the Brookhaven National Laboratory, supported by the US Department of Energy, for assistance with data collection. We thank Z. Wang for assistance with X-ray data collection at the APS.

Author Contributions Y.W. and G.S. expressed and purified wild-type T. thermophilus Ago and its catalytic mutants, and also grew crystals of the various ternary complexes. H.L. collected X-ray diffraction data on the various NE-CAT beam lines, and Y.W. solved the structures of these ternary complexes. D.J.P. supervised the structural studies. S.J. was responsible for the cleavage assays on Ago with modified DNA and RNA target strands, and G.S.W. purified the phosphorothioate diastereomers and quality controlled oligonucleotides, under the supervision of T.T. D.J.P. and T.T. were primarily responsible for writing the structural and biochemical contents of the paper, respectively, and all authors read and approved the submitted manuscript.

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Correspondence to Thomas Tuschl or Dinshaw J. Patel.

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Competing interests

[COMPETING INTERESTS: T.T. is a cofounder and scientific advisor to Alnylam Pharmaceuticals and an advisor to Regulus Therapeutics.]

Supplementary information

Supplementary Information

This file contains Supplementary Notes, Supplementary Tables 1-4 and Supplementary Figures 1-27 with Legends. (PDF 10468 kb)

Supplementary Movie 1

This movie shows interconversion between Ago binary complex containing guide DNA (in red) and Ago ternary complex containing added 12-nucleotide target RNA (in blue). (AVI 3932 kb)

Supplementary Movie 2

This movie shows interconversion between Ago ternary complexes containing guide DNA (in red) and added 12- and 15-nucleotide target RNAs (in blue). (AVI 3704 kb)

Supplementary Movie 3

This movie shows rotation of Ago ternary complex structure containing guide DNA (in red) and 19-nucleotide target RNA (in blue). (AVI 13088 kb)

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