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Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs

Nature Neuroscience volume 15, pages 1488–1497 (2012)Cite this article

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Abstract

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell–derived human neurons and in TDP-43 aggregate–containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.

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Figure 1: FUS/TLS RNA targets in mouse and human brain.
Figure 2: FUS/TLS binding patterns in mouse and human brain.
Figure 3: Changes in the expression of FUS/TLS and TDP-43 targets after depletion (knockdown, KD) of either FUS/TLS or TDP-43 in brain and spinal cord.
Figure 4: FUS/TLS-dependent alternative splicing in mouse brain.
Figure 5: Alterations of TDP-43 and FUS/TLS human targets in neurons derived from ES cells.
Figure 6: Reduction of TDP-43 and FUS/TLS RNA targets in motor neurons from sporadic ALS patients.

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References

  1. Da Cruz, S. & Cleveland, D.W. Understanding the role of TDP-43 and FUS/TLS in ALS and beyond. Curr. Opin. Neurobiol. 21, 904–919 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Neumann, M. et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130–133 (2006).

    CAS  PubMed  Google Scholar 

  3. Arai, T. et al. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 351, 602–611 (2006).

    CAS  PubMed  Google Scholar 

  4. Polymenidou, M. et al. Misregulated RNA processing in amyotrophic lateral sclerosis. Brain Res. 1462, 3–15 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Gitcho, M.A. et al. TDP-43 A315T mutation in familial motor neuron disease. Ann. Neurol. 63, 535–538 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Kabashi, E. et al. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nat. Genet. 40, 572–574 (2008).

    CAS  PubMed  Google Scholar 

  7. Sreedharan, J. et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668–1672 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Kwiatkowski, T.J. et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323, 1205–1208 (2009).

    CAS  PubMed  Google Scholar 

  9. Vance, C. et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323, 1208–1211 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Neumann, M. et al. A new subtype of frontotemporal lobar degeneration with FUS pathology. Brain 132, 2922–2931 (2009).

    PubMed  PubMed Central  Google Scholar 

  11. Woulfe, J., Gray, D.A. & Mackenzie, I.R. FUS-immunoreactive intranuclear inclusions in neurodegenerative disease. Brain Pathol. 20, 589–597 (2010).

    CAS  PubMed  Google Scholar 

  12. Munoz, D.G. et al. FUS pathology in basophilic inclusion body disease. Acta Neuropathol. 118, 617–627 (2009).

    CAS  PubMed  Google Scholar 

  13. Mackenzie, I.R. et al. Pathological heterogeneity in amyotrophic lateral sclerosis with FUS mutations: two distinct patterns correlating with disease severity and mutation. Acta Neuropathol. 122, 87–98 (2011).

    PubMed  PubMed Central  Google Scholar 

  14. Lagier-Tourenne, C., Polymenidou, M. & Cleveland, D.W. TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum. Mol. Genet. 19, R46–R64 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Polymenidou, M. et al. Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Nat. Neurosci. 14, 459–468 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Tollervey, J.R. et al. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat. Neurosci. 14, 452–458 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Huelga, S.C. et al. Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins. Cell Rep. 1, 167–178 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Lerga, A. et al. Identification of an RNA binding specificity for the potential splicing factor TLS. J. Biol. Chem. 276, 6807–6816 (2001).

    CAS  PubMed  Google Scholar 

  19. Fujii, R. et al. The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology. Curr. Biol. 15, 587–593 (2005).

    CAS  PubMed  Google Scholar 

  20. Ling, S.C. et al. ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS. Proc. Natl. Acad. Sci. USA 107, 13318–13323 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhang, C. et al. Integrative modeling defines the Nova splicing-regulatory network and its combinatorial controls. Science 329, 439–443 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ameur, A. et al. Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain. Nat. Struct. Mol. Biol. 18, 1435–1440 (2011).

    CAS  PubMed  Google Scholar 

  23. Smith, R.A. et al. Antisense oligonucleotide therapy for neurodegenerative disease. J. Clin. Invest. 116, 2290–2296 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Kordasiewicz, H.B. et al. Sustained therapeutic reversal of Huntington's disease by transient repression of Huntingtin synthesis. Neuron 74, 1031–1044 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Colombrita, C. et al. TDP-43 and FUS RNA-binding proteins bind distinct sets of cytoplasmic messenger RNAs and differently regulate their post-transcriptional fate in motoneuron-like cells. J. Biol. Chem. 287, 15635–15647 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Ishigaki, S. et al. Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions. Sci. Rep. 2, 529 (2012).

    PubMed  PubMed Central  Google Scholar 

  27. Rogelj, B. et al. Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain. Sci. Rep. 2, 603 (2012).

    PubMed  PubMed Central  Google Scholar 

  28. Hicks, G.G. et al. Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death. Nat. Genet. 24, 175–179 (2000).

    CAS  PubMed  Google Scholar 

  29. Okuda, T., Kokame, K. & Miyata, T. Differential expression patterns of NDRG family proteins in the central nervous system. J. Histochem. Cytochem. 56, 175–182 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Mitchelmore, C. et al. NDRG2: a novel Alzheimer's disease associated protein. Neurobiol. Dis. 16, 48–58 (2004).

    CAS  PubMed  Google Scholar 

  31. Hutton, M. et al. Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393, 702–705 (1998).

    CAS  PubMed  Google Scholar 

  32. Liu, B.P., Fournier, A., GrandPre, T. & Strittmatter, S.M. Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science 297, 1190–1193 (2002).

    CAS  PubMed  Google Scholar 

  33. Rabin, S.J. et al. Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology. Hum. Mol. Genet. 19, 313–328 (2010).

    CAS  PubMed  Google Scholar 

  34. Bodansky, A. et al. TDP-43 and ubiquitinated cytoplasmic aggregates in sporadic ALS are low frequency and widely distributed in the lower motor neuron columns independent of disease spread. Amyotroph. Lateral Scler. 11, 321–327 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Hoell, J.I. et al. RNA targets of wild-type and mutant FET family proteins. Nat. Struct. Mol. Biol. 18, 1428–1431 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Kitada, T. et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605–608 (1998).

    CAS  PubMed  Google Scholar 

  37. Itier, J.M. et al. Parkin gene inactivation alters behavior and dopamine neurotransmission in the mouse. Hum. Mol. Genet. 12, 2277–2291 (2003).

    CAS  PubMed  Google Scholar 

  38. Goldberg, M.S. et al. Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons. J. Biol. Chem. 278, 43628–43635 (2003).

    CAS  PubMed  Google Scholar 

  39. Morohashi, Y. et al. Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4. J. Biol. Chem. 277, 14965–14975 (2002).

    CAS  PubMed  Google Scholar 

  40. Massone, S. et al. RNA polymerase III drives alternative splicing of the potassium channel-interacting protein contributing to brain complexity and neurodegeneration. J. Cell Biol. 193, 851–866 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Lin, S., Coutinho-Mansfield, G., Wang, D., Pandit, S. & Fu, X.D. The splicing factor SC35 has an active role in transcriptional elongation. Nat. Struct. Mol. Biol. 15, 819–826 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Wada, Y. et al. A wave of nascent transcription on activated human genes. Proc. Natl. Acad. Sci. USA 106, 18357–18361 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Trcek, T., Larson, D.R., Moldon, A., Query, C.C. & Singer, R.H. Single-molecule mRNA decay measurements reveal promoter-regulated mRNA stability in yeast. Cell 147, 1484–1497 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Tan, A.Y., Riley, T.R., Coady, T., Bussemaker, H.J. & Manley, J.L. TLS/FUS (translocated in liposarcoma/fused in sarcoma) regulates target gene transcription via single-stranded DNA response elements. Proc. Natl. Acad. Sci. USA 109, 6030–6035 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Bertolotti, A. et al. EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: interactions between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes. Mol. Cell Biol. 18, 1489–1497 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang, X. et al. Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription. Nature 454, 126–130 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Ticozzi, N. et al. Mutational analysis reveals the FUS homolog TAF15 as a candidate gene for familial amyotrophic lateral sclerosis. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 156B, 285–290 (2011).

    CAS  PubMed  Google Scholar 

  48. Couthouis, J. et al. A yeast functional screen predicts new candidate ALS disease genes. Proc. Natl. Acad. Sci. USA 108, 20881–20890 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Couthouis, J. et al. Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis. Hum. Mol. Genet. 21, 2899–2911 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Neumann, M. et al. FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations. Brain 134, 2595–2609 (2011).

    PubMed  PubMed Central  Google Scholar 

  51. Parkhomchuk, D. et al. Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res. 37, e123 (2009).

    PubMed  PubMed Central  Google Scholar 

  52. Zhang, C. & Darnell, R.B. Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data. Nat. Biotechnol. 29, 607–614 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Yeo, G.W. et al. An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat. Struct. Mol. Biol. 16, 130–137 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Zisoulis, D.G. et al. Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nat. Struct. Mol. Biol. 17, 173–179 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    CAS  PubMed  Google Scholar 

  57. Sugnet, C.W. et al. Unusual intron conservation near tissue-regulated exons found by splicing microarrays. PLOS Comput. Biol. 2, e4 (2006).

    PubMed  PubMed Central  Google Scholar 

  58. Yeo, G.W. et al. Alternative splicing events identified in human embryonic stem cells and neural progenitors. PLOS Comput. Biol. 3, 1951–1967 (2007).

    CAS  PubMed  Google Scholar 

  59. Marchetto, M.C. et al. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143, 527–539 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Brooks, B.R., Miller, R.G., Swash, M. & Munsat, T.L. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 1, 293–299 (2000).

    CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank members of B. Ren's laboratory, especially Z. Ye, S. Kuan, U. Wagner and L. Edsall, for technical help with the Illumina sequencing, M. Ares Jr. for generous support, as well as the members of the Yeo and Cleveland laboratories and the team at Isis Pharmaceuticals for critical comments and suggestions on this project. This work was supported by grants from the US National Institutes of Health (R37NS27036 to D.W.C. and K99NS075216 to M.P.). M.P. is the recipient of a long-term fellowship from the international Human Frontier Science Program Organization. C.L.-T. is the recipient of a Career Development Award from the Muscular Dystrophy Association and the Milton-Safenowitz post-doctoral fellowship from the Amyotrophic Lateral Sclerosis Association. D.W.C. receives salary support from the Ludwig Institute for Cancer Research. S.C.H. is funded by a US National Science Foundation Graduate Research Fellowship. This work was also supported by grant number R01NS075449 from the US National Institute of Neurological Disorders and Stroke, and was partially supported by grants from the US National Institutes of Health (HG004659 and GM084317) and the California Institute for Regenerative Medicine (RB1-01413 and RB3-05009) to G.W.Y. G.W.Y. is a recipient of the Alfred P. Sloan Research Fellowship.

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  1. Clotilde Lagier-Tourenne, Magdalini Polymenidou and Kasey R Hutt: These authors contributed equally to this work.

Authors and Affiliations

  1. Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, California, USA

    Clotilde Lagier-Tourenne, Magdalini Polymenidou, Kevin M Clutario, Shuo-Chien Ling & Don W Cleveland

  2. Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA

    Clotilde Lagier-Tourenne, Magdalini Polymenidou, Kasey R Hutt, Anthony Q Vu, Stephanie C Huelga, Kevin M Clutario, Shuo-Chien Ling, Tiffany Y Liang, Don W Cleveland & Gene W Yeo

  3. Stem Cell Program and Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA

    Kasey R Hutt, Anthony Q Vu, Stephanie C Huelga, Tiffany Y Liang & Gene W Yeo

  4. Department of Neurosciences, University of California at San Diego, La Jolla, California, USA

    Michael Baughn & John Ravits

  5. Isis Pharmaceuticals, Carlsbad, California, USA

    Curt Mazur, Edward Wancewicz, Aneeza S Kim, Andy Watt, Sue Freier & C Frank Bennett

  6. MB Institute of Cell Biology, University of Manitoba, Winnipeg, Canada

    Geoffrey G Hicks

  7. Department of Molecular, RNA Center, Cell and Developmental Biology, Sinsheimer Labs, University of California, Santa Cruz, California, USA

    John Paul Donohue & Lily Shiue

  8. A*STAR and Department of Biological Sciences, Molecular Engineering Laboratory, National University of Singapore, Singapore

    Gene W Yeo

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Contributions

C.L.-T., M.P., A.Q.V., M.B. and K.M.C. performed the experiments. K.R.H., S.C.H. and T.Y.L. conducted the bioinformatics analysis. S.-C.L. developed the polyclonal FUS/TLS-specific antibody Ab3. J.P.D. and L.S. conducted the preliminary splice-junction microarray analyses. G.G.H. provided the brain samples from the Fus/Tls−/− mice. M.P., C.L.-T., C.M., E.W., A.S.K., A.W., S.F. and C.F.B. conducted the ASO experiments. J.R. provided the ALS patient tissues. C.L.-T., M.P., K.R.H., G.W.Y. and D.W.C. designed the experiments. C.L.-T., M.P., K.R.H., A.Q.V., G.W.Y. and D.W.C. wrote the manuscript.

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Correspondence to Don W Cleveland or Gene W Yeo.

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Lagier-Tourenne, C., Polymenidou, M., Hutt, K. et al. Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs. Nat Neurosci 15, 1488–1497 (2012). https://doi.org/10.1038/nn.3230

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