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CLP1 links tRNA metabolism to progressive motor-neuron loss

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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.

References

  1. Weitzer, S. & Martinez, J. The human RNA kinase hClp1 is active on 3′ transfer RNA exons and short interfering RNAs. Nature 447, 222–226 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  2. Ramirez, A., Shuman, S. & Schwer, B. Human RNA 5′-kinase (hClp1) can function as a tRNA splicing enzyme in vivo . RNA 14, 1737–1745 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Jain, R. & Shuman, S. Characterization of a thermostable archaeal polynucleotide kinase homologous to human Clp1. RNA 15, 923–931 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. de Vries, H. et al. Human pre-mRNA cleavage factor IIm contains homologs of yeast proteins and bridges two other cleavage factors. EMBO J. 19, 5895–5904 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Minvielle-Sebastia, L., Preker, P. J., Wiederkehr, T., Strahm, Y. & Keller, W. The major yeast poly(A)-binding protein is associated with cleavage factor IA and functions in premessenger RNA 3′-end formation. Proc. Natl Acad. Sci. USA 94, 7897–7902 (1997)

    Article  ADS  CAS  PubMed  Google Scholar 

  6. Holbein, S. et al. The P-loop domain of yeast Clp1 mediates interactions between CF IA and CPF factors in pre-mRNA 3′ end formation. PLoS ONE 6, e29139 (2011)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Haddad, R. et al. An essential role for Clp1 in assembly of polyadenylation complex CF IA and Pol II transcription termination. Nucleic Acids Res. 40, 1226–1239 (2012)

    Article  CAS  PubMed  Google Scholar 

  8. Ghazy, M. A. et al. The interaction of Pcf11 and Clp1 is needed for mRNA 3′-end formation and is modulated by amino acids in the ATP-binding site. Nucleic Acids Res. 40, 1214–1225 (2012)

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Paushkin, S. V., Patel, M., Furia, B. S., Peltz, S. W. & Trotta, C. R. Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3′ end formation. Cell 117, 311–321 (2004)

    Article  CAS  PubMed  Google Scholar 

  10. Trotta, C. R. et al. The yeast tRNA splicing endonuclease: a tetrameric enzyme with two active site subunits homologous to the archaeal tRNA endonucleases. Cell 89, 849–858 (1997)

    Article  CAS  PubMed  Google Scholar 

  11. Zillmann, M., Gorovsky, M. A. & Phizicky, E. M. Conserved mechanism of tRNA splicing in eukaryotes. Mol. Cell. Biol. 11, 5410–5416 (1991)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhao, C. et al. Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bβ. Cell 105, 587–597 (2001)

    Article  CAS  PubMed  Google Scholar 

  13. Sánchez-Carbente, M. R., Castro-Obregon, S., Covarrubias, L. & Narvaez, V. Motoneuronal death during spinal cord development is mediated by oxidative stress. Cell Death Differ. 12, 279–291 (2005)

    Article  PubMed  Google Scholar 

  14. Medana, I. M. & Esiri, M. M. Axonal damage: a key predictor of outcome in human CNS diseases. Brain 126, 515–530 (2003)

    Article  CAS  PubMed  Google Scholar 

  15. Atkin, J. D. et al. Properties of slow- and fast-twitch muscle fibres in a mouse model of amyotrophic lateral sclerosis. Neuromuscul. Disord. 15, 377–388 (2005)

    Article  PubMed  Google Scholar 

  16. Yamasaki, S., Ivanov, P., Hu, G. F. & Anderson, P. Angiogenin cleaves tRNA and promotes stress-induced translational repression. J. Cell Biol. 185, 35–42 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ivanov, P., Emara, M. M., Villen, J., Gygi, S. P. & Anderson, P. Angiogenin-induced tRNA fragments inhibit translation initiation. Mol. Cell 43, 613–623 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Son, E. Y. et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lambert, P. F., Kashanchi, F., Radonovich, M. F., Shiekhattar, R. & Brady, J. N. Phosphorylation of p53 serine 15 increases interaction with CBP. J. Biol. Chem. 273, 33048–33053 (1998)

    Article  CAS  PubMed  Google Scholar 

  20. Dumaz, N. & Meek, D. W. Serine 15 phosphorylation stimulates p53 transactivation but does not directly influence interaction with HDM2. EMBO J. 18, 7002–7010 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chao, C. et al. Cell type- and promoter-specific roles of Ser18 phosphorylation in regulating p53 responses. J. Biol. Chem. 278, 41028–41033 (2003)

    Article  CAS  PubMed  Google Scholar 

  22. Arber, S. et al. Requirement for the homeobox gene Hb9 in the consolidation of motor neuron identity. Neuron 23, 659–674 (1999)

    Article  CAS  PubMed  Google Scholar 

  23. Hanada, R. et al. Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway. Nature Med. 10, 1067–1073 (2004)

    Article  CAS  PubMed  Google Scholar 

  24. Tuck, A. C. & Tollervey, D. RNA in pieces. Trends Genet. 27, 422–432 (2011)

    Article  CAS  PubMed  Google Scholar 

  25. Hurto, R. L. Unexpected functions of tRNA and tRNA processing enzymes. Adv. Exp. Med. Biol. 722, 137–155 (2011)

    Article  CAS  PubMed  Google Scholar 

  26. Lee, Y. S., Shibata, Y., Malhotra, A. & Dutta, A. A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). Genes Dev. 23, 2639–2649 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Herbst, R., Iskratsch, T., Unger, E. & Bittner, R. E. Aberrant development of neuromuscular junctions in glycosylation-defective Largemyd mice. Neuromuscul. Disord. 19, 366–378 (2009)

    Article  PubMed  PubMed Central  Google Scholar 

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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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Authors and Affiliations

Authors

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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