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Multimeric assembly and biochemical characterization of the Trax–translin endonuclease complex

Nature Structural & Molecular Biology volume 18, pages 658664 (2011) | Download Citation

Abstract

Trax–translin heteromers, also known as C3PO, have been proposed to activate the RNA-induced silencing complex (RISC) by facilitating endonucleolytic cleavage of the siRNA passenger strand. We report on the crystal structure of hexameric Drosophila C3PO formed by truncated translin and Trax, along with electron microscopic and mass spectrometric studies on octameric C3PO formed by full-length translin and Trax. Our studies establish that Trax adopts the translin fold, possesses catalytic centers essential for C3PO's endoRNase activity and interacts extensively with translin to form an octameric assembly. The catalytic pockets of Trax subunits are located within the interior chamber of the octameric scaffold. Truncated C3PO, like full-length C3PO, shows endoRNase activity that leaves 3′-hydroxyl–cleaved ends. We have measured the catalytic activity of C3PO and shown it to cleave almost stoichiometric amounts of substrate per second.

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Acknowledgements

We thank H. Li from the Sloan-Kettering Institute for assistance with synchrotron data collection, H. Wu from Weill Cornell Medical College for access to and assistance with MALS experiments, and P. Upla from the New York Structural Biology Center for help with the electron microscopy of labeled proteins. We are grateful to the staff of the X-29 beamline at the National Synchrotron Light Source, Brookhaven National Laboratory, and the staff of the ID-24-E beamline at the Advanced Photon Source, Argonne National Laboratory, for their help with data collection. D.J.P. is supported by US National Institutes of Health (NIH) grant AI068776. T.T. is supported by funds from the Howard Hughes Medical Institute and the NIH. D.J.P. and T.T. were jointly supported by the Starr Cancer Consortium. A.Y.P. and C.V.R. acknowledge funding from the Biotechnology and Biological Sciences Research Council and the Royal Society.

Author information

Author notes

    • Yuan Tian
    • , Dhirendra K Simanshu
    •  & Manuel Ascano

    These authors contributed equally to this work.

Affiliations

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

    • Yuan Tian
    • , Dhirendra K Simanshu
    •  & Dinshaw J Patel
  2. Graduate Program in Neuroscience, Weill Medical College of Cornell University, New York, New York, USA.

    • Yuan Tian
  3. The Rockefeller University, New York, New York, USA.

    • Manuel Ascano
    • , Stefan A Juranek
    •  & Thomas Tuschl
  4. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, New York, New York, USA.

    • Manuel Ascano
    •  & Thomas Tuschl
  5. New York Structural Biology Center, New York, New York, USA.

    • Ruben Diaz-Avalos
    •  & William J Rice
  6. Department of Chemistry, University of Oxford, Oxford, UK.

    • Ah Young Park
    •  & Carol V Robinson
  7. Department of Biochemistry, Weill Medical College of Cornell University, New York, USA.

    • Qian Yin

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Contributions

Y.T. designed and conducted the experiments leading to crystallization of C3PO and undertook initial structural characterization, and D.K.S. improved the density map, built the model and finished the refinement under the supervision of D.J.P.; M.A. and S.A.J. did the cleavage assays under the supervision of T.T.; R.D.-A. and W.J.R. conducted the EM studies; A.Y.P. conducted the MS studies under the supervision of C.V.R.; Q.Y. and Y.T. conducted the MALS studies. All authors participated in writing the paper.

Competing interests

TT is a cofounder and scientific advisor to Alnylam Pharmaceuticals and an advisor to Regulus Therapeutics.

Corresponding authors

Correspondence to Thomas Tuschl or Dinshaw J Patel.

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https://doi.org/10.1038/nsmb.2069

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