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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Carthew, R.W. & Sontheimer, E.J. Origins and mechanisms of miRNAs and siRNAs. Cell 136, 642–655 (2009).
Kim, V.N., Han, J. & Siomi, M.C. Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell Biol. 10, 126–139 (2009).
Liu, Q. et al. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301, 1921–1925 (2003).
Tomari, Y., Matranga, C., Haley, B., Martinez, N. & Zamore, P.D. A protein sensor for siRNA asymmetry. Science 306, 1377–1380 (2004).
Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D.J. Structure of the guide-strand-containing argonaute silencing complex. Nature 456, 209–213 (2008).
Kawamata, T. & Tomari, Y. Making RISC. Trends Biochem. Sci. 35, 368–376 (2010).
Wang, Y. et al. Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes. Nature 461, 754–761 (2009).
Liu, Y. et al. C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation. Science 325, 750–753 (2009).
Jaendling, A. & McFarlane, R.J. Biological roles of translin and translin-associated factor-X: RNA metabolism comes to the fore. Biochem. J. 429, 225–234 (2010).
Li, Z., Wu, Y. & Baraban, J.M. The Translin/Trax RNA binding complex: clues to function in the nervous system. Biochim. Biophys. Acta 1779, 479–485 (2008).
Eliahoo, E. et al. Mapping of interaction sites of the Schizosaccharomyces pombe protein Translin with nucleic acids and proteins: a combined molecular genetics and bioinformatics study. Nucleic Acids Res. 38, 2975–2989 (2010).
Lluis, M., Hoe, W., Schleit, J. & Robertus, J. Analysis of nucleic acid binding by a recombinant translin–trax complex. Biochem. Biophys. Res. Commun. 396, 709–713 (2010).
Gupta, G.D., Makde, R.D., Rao, B.J. & Kumar, V. Crystal structures of Drosophila mutant translin and characterization of translin variants reveal the structural plasticity of translin proteins. FEBS J. 275, 4235–4249 (2008).
Pascal, J.M., Hart, P.J., Hecht, N.B. & Robertus, J.D. Crystal structure of TB-RBP, a novel RNA-binding and regulating protein. J. Mol. Biol. 319, 1049–1057 (2002).
Sugiura, I. et al. Structure of human translin at 2.2 A resolution. Acta Crystallogr. D Biol. Crystallogr. 60, 674–679 (2004).
VanLoock, M.S., Yu, X., Kasai, M. & Egelman, E.H. Electron microscopic studies of the translin octameric ring. J. Struct. Biol. 135, 58–66 (2001).
Claussen, M., Koch, R., Jin, Z.Y. & Suter, B. Functional characterization of Drosophila Translin and Trax. Genetics 174, 1337–1347 (2006).
Yang, W. An equivalent metal ion in one- and two-metal-ion catalysis. Nat. Struct. Mol. Biol. 15, 1228–1231 (2008).
Aharoni, A., Baran, N. & Manor, H. Characterization of a multisubunit human protein which selectively binds single stranded d(GA)n and d(GT)n sequence repeats in DNA. Nucleic Acids Res. 21, 5221–5228 (1993).
Aoki, K., Suzuki, K., Ishida, R. & Kasai, M. The DNA binding activity of Translin is mediated by a basic region in the ring-shaped structure conserved in evolution. FEBS Lett. 443, 363–366 (1999).
Aoki, K. et al. A novel gene, Translin, encodes a recombination hotspot binding protein associated with chromosomal translocations. Nat. Genet. 10, 167–174 (1995).
Gupta, G.D. et al. Co-expressed recombinant human Translin-Trax complex binds DNA. FEBS Lett. 579, 3141–3146 (2005).
Kasai, M. et al. The translin ring specifically recognizes DNA ends at recombination hot spots in the human genome. J. Biol. Chem. 272, 11402–11407 (1997).
Laufman, O., Ben Yosef, R., Adir, N. & Manor, H. Cloning and characterization of the Schizosaccharomyces pombe homologs of the human protein Translin and the Translin-associated protein TRAX. Nucleic Acids Res. 33, 4128–4139 (2005).
Sengupta, K. & Rao, B.J. Translin binding to DNA: recruitment through DNA ends and consequent conformational transitions. Biochemistry 41, 15315–15326 (2002).
Wang, J., Boja, E.S., Oubrahim, H. & Chock, P.B. Testis brain ribonucleic acid-binding protein/translin possesses both single-stranded and double-stranded ribonuclease activities. Biochemistry 43, 13424–13431 (2004).
Wu, X.Q., Gu, W., Meng, X. & Hecht, N.B. The RNA-binding protein, TB-RBP, is the mouse homologue of translin, a recombination protein associated with chromosomal translocations. Proc. Natl. Acad. Sci. USA 94, 5640–5645 (1997).
Wu, X.Q., Xu, L. & Hecht, N.B. Dimerization of the testis brain RNA-binding protein (translin) is mediated through its C-terminus and is required for DNA- and RNA-binding. Nucleic Acids Res. 26, 1675–1680 (1998).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. & Paciorek, W. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D Biol. Crystallogr. 59, 2023–2030 (2003).
Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 103, 8060–8065 (2006).
Sobott, F., Hernandez, H., McCammon, M.G., Tito, M.A. & Robinson, C.V. A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. Anal. Chem. 74, 1402–1407 (2002).
Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Alefelder, S., Patel, B.K. & Eckstein, F. Incorporation of terminal phosphorothioates into oligonucleotides. Nucleic Acids Res. 26, 4983–4988 (1998).
Akbergenov, R. et al. Molecular characterization of geminivirus-derived small RNAs in different plant species. Nucleic Acids Res. 34, 462–471 (2006).
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
Authors and Affiliations
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.
Corresponding authors
Ethics declarations
Competing interests
TT is a cofounder and scientific advisor to Alnylam Pharmaceuticals and an advisor to Regulus Therapeutics.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–9 and Supplementary Methods (PDF 2249 kb)
Rights and permissions
About this article
Cite this article
Tian, Y., Simanshu, D., Ascano, M. et al. Multimeric assembly and biochemical characterization of the Trax–translin endonuclease complex. Nat Struct Mol Biol 18, 658–664 (2011). https://doi.org/10.1038/nsmb.2069
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.2069
This article is cited by
-
Translin: A multifunctional protein involved in nucleic acid metabolism
Journal of Biosciences (2019)
-
Long non-coding RNAs: new players in ocular neovascularization
Molecular Biology Reports (2014)
-
Structural basis for duplex RNA recognition and cleavage by Archaeoglobus fulgidus C3PO
Nature Structural & Molecular Biology (2013)
-
The translin–TRAX complex (C3PO) is a ribonuclease in tRNA processing
Nature Structural & Molecular Biology (2012)
-
The N domain of Argonaute drives duplex unwinding during RISC assembly
Nature Structural & Molecular Biology (2012)