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CLP1 links tRNA metabolism to progressive motor-neuron loss

Nature volume 495, pages 474–480 (2013)Cite this article

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Abstract

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1K/K ) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1K/K mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.

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Figure 1: Respiratory failure and impaired innervation of the diaphragm.
Figure 2: Viable Clp1 K/K embryos develop neuromuscular atrophy.
Figure 3: Progressive loss of lower motor neurons.
Figure 4: Identification of a novel tRNA fragment.
Figure 5: Neonatal lethality and motor-neuron loss are mediated by p53.
Figure 6: CLP1 acts in motor neurons.

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Gene Expression Omnibus

Data deposits

Data have been deposited in the Gene Expression Omnibus under accession numbers GSE35924 and GSE39275.

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Acknowledgements

We thank A. Meixner, M. Foong, T. Nakashima, H. C. Theussl, J. R. Wojciechowski, A. Bichl, the mouse pathology unit of the Universitätsklinikum Hamburg-Eppendorf, and G. P. Resch for discussions and technical support. We also thank T. Buerckstuemmer for providing the pRV-NTAP vector. J.M.P. is supported by grants from the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), the Austrian Ministry of Sciences, the Austrian Academy of Sciences, AustroMouse network of Genome Research in Austria (GEN-AU), Apoptosis systems biology applied to cancer and AIDS (ApoSys) and a European Research Council Advanced Grant from the European Union. J.M., S.W. and B.M. are supported by IMBA and GEN-AU (AustroMouse). T.H. is supported by the Japan Society for the Promotion of Science and the Astellas Foundation. J.K.I. was supported by National Institutes of Health (NIH) grant K99NS077435-01A1. M.G. is supported by grants from the German Research Foundation (DFG) (FG885 and GRK 1459), the Landesexzellenzinitiative Hamburg (Neurodapt). R.H. was supported by the Austrian Science Fund (P19223, P21667). C.J.W. is supported by the NIH (NS038253).

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Author notes

  1. Toshikatsu Hanada and Stefan Weitzer: These authors contributed equally to this work.

Authors and Affiliations

  1. IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna 1030, Austria ,

    Toshikatsu Hanada, Stefan Weitzer, Barbara Mair, Reiko Hanada, Michael Orthofer, Vukoslav Komnenovic, Javier Martinez & Josef M. Penninger

  2. Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany ,

    Christian Bernreuther & Markus Glatzel

  3. Program in Neurobiology and F. M. Kirby Neurobiology Center, Boston Children’s Hospital, Boston, 02115, Massachusetts, USA

    Brian J. Wainger, Shane J. Cronin & Clifford J. Woolf

  4. Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA

    Brian J. Wainger

  5. Department of Stem Cell and Regenerative Biology, Howard Hughes Medical Institute, Harvard Stem Cell Institute. Boston, 02115, Massachusetts, USA

    Justin Ichida & Kevin C. Eggan

  6. Department of Biological Chemistry. The Weizmann Institute of Science, Rehovot 76100, Israel,

    Adi Minis & Avraham Yaron

  7. Department of Urology, Oita University Faculty of Medicine, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita 879-5593, Japan,

    Fuminori Sato & Hiromitsu Mimata

  8. Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan,

    Akihiko Yoshimura

  9. Campus Science Support Facilities GmbH, Dr. Bohr-Gasse 3, Vienna 1030, Austria ,

    Ido Tamir

  10. Division Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, Innsbruck 6020, Austria,

    Johannes Rainer & Reinhard Kofler

  11. Department of Neurobiology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Clifford J. Woolf

  12. Center for Brain Research, Medical University of Vienna, Spitalgasse 4, Vienna 1090, Austria ,

    Ruth Herbst

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  1. Toshikatsu Hanada
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Contributions

T.H. generated mutant mice, performed mouse phenotyping and developed cell lines with help from R.H. S.W. and B.M. performed all biochemical assays and northern blots. V.K. performed histological analysis. F.S., H.M., S.J.C. and A.Y. provided key reagents and technical help for generation of mutant mice. I.T. analysed tRNA fragment distributions. M.O. performed calorimetric experiments. B.J.W., J.I., K.C.E. and C.J.W. performed and helped with transdifferentiation experiments and patch clamping. R.H. performed immunostainings and assessment of NMJs and motor-neuron pathfinding. A.M. and A.Y. performed DRG explant cultures, and J.R. and R.K. carried out exon arrays. C.B. and M.G. performed histological and immunohistochemical staining of, and assessed, peripheral nerve and brain structures. J.M. and J.M.P. coordinated the project and wrote the manuscript.

Corresponding authors

Correspondence to Javier Martinez or Josef M. Penninger.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-33. (PDF 19765 kb)

Supplementary Information

This file contains Supplementary Methods and additional references. (PDF 320 kb)

3 months old Clp1+/+ mouse (CBA/J)

This video shows a 3 months old control Clp1+/+ mouse on a CBA/J background. The mouse displays normal gait. (MOV 11495 kb)

3 months old Clp1K/K mouse (CBA/J)

This video shows a 3 months old Clp1K/K mouse on a CBA/J background. The mouse displays motor ataxia. (MOV 19268 kb)

12 months old Clp1+/+ mouse (CBA/J)

This video shows a 12 months old control Clp1+/+ mouse on a CBA/J background. The mouse displays normal gait. (MOV 9246 kb)

12 months old Clp1K/K mouse (CBA/J)

This video shows a 12 months old Clp1K/K mouse on a CBA/J background. The mouse displays severely impaired motor function and limb paralysis. (MOV 12751 kb)

12 months old Clp1K/K mouse (CBA/J)

This video shows a 12 months old Clp1K/K mouse on a CBA/J background. The mouse displays severely impaired motor function and limb paralysis. (MOV 22376 kb)

12 months old Clp1K/K mouse (CBA/J)

This video shows a 12 months old Clp1K/K mouse on a CBA/J background. The mouse displays severely impaired motor function and limb paralysis. (MOV 18474 kb)

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Hanada, T., Weitzer, S., Mair, B. et al. CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature 495, 474–480 (2013). https://doi.org/10.1038/nature11923

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