Advertisement

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*

  • Claudia Colombrita
    Affiliations
    Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 20149, Italy,
    Search for articles by this author
  • Elisa Onesto
    Footnotes
    Affiliations
    Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 20149, Italy,
    Search for articles by this author
  • Francesca Megiorni
    Affiliations
    Department of Experimental Medicine, Sapienza University of Rome, Rome 00161, Italy,
    Search for articles by this author
  • Antonio Pizzuti
    Affiliations
    Department of Experimental Medicine, Sapienza University of Rome, Rome 00161, Italy,
    Search for articles by this author
  • Francisco E. Baralle
    Affiliations
    International Centre for Genetic Engineering and Biotechnology, AREA Science Park, Trieste 34149, Italy, and
    Search for articles by this author
  • Emanuele Buratti
    Affiliations
    International Centre for Genetic Engineering and Biotechnology, AREA Science Park, Trieste 34149, Italy, and
    Search for articles by this author
  • Vincenzo Silani
    Affiliations
    Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 20149, Italy,

    Department of Neurological Sciences, “Dino Ferrari” Center, Università degli Studi di Milano, Milan 20122, Italy
    Search for articles by this author
  • Antonia Ratti
    Correspondence
    To whom correspondence should be addressed: Dept. of Neurology, IRCCS Istituto Auxologico Italiano, Via Zucchi, 18–20095 Cusano Milanino, Milan, Italy. Tel.: 39-02-619113045; Fax: 39-02-619113033
    Affiliations
    Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 20149, Italy,

    Department of Neurological Sciences, “Dino Ferrari” Center, Università degli Studi di Milano, Milan 20122, Italy
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the Italian Agency of Research on ALS (AriSLA) (Grant RBPALS) and Fondazione CARIPLO (Grant 2008-2307).
    This article contains supplemental Tables 1–7 and Figs. 1–5.
    1 Both authors contributed equally to this work.
Open AccessPublished:March 16, 2012DOI:https://doi.org/10.1074/jbc.M111.333450
      The RNA-binding proteins TDP-43 and FUS form abnormal cytoplasmic aggregates in affected tissues of patients with amyotrophic lateral sclerosis and frontotemporal lobar dementia. TDP-43 and FUS localize mainly in the nucleus where they regulate pre-mRNA splicing, but they are also involved in mRNA transport, stability, and translation. To better investigate their cytoplasmic activities, we applied an RNA immunoprecipitation and chip analysis to define the mRNAs associated to TDP-43 and FUS in the cytoplasmic ribonucleoprotein complexes from motoneuronal NSC-34 cells. We found that they bind different sets of mRNAs although converging on common cellular pathways. Bioinformatics analyses identified the (UG)n consensus motif in 80% of 3′-UTR sequences of TDP-43 targets, whereas for FUS the binding motif was less evident. By in vitro assays we validated binding to selected target 3′-UTRs, including Vegfa and Grn for TDP-43, and Vps54, Nvl, and Taf15 for FUS. We showed that TDP-43 has a destabilizing activity on Vegfa and Grn mRNAs and may ultimately affect progranulin protein content, whereas FUS does not affect mRNA stability/translation of its targets. We also demonstrated that three different point mutations in TDP-43 did not change the binding affinity for Vegfa and Grn mRNAs or their protein level. Our data indicate that TDP-43 and FUS recognize distinct sets of mRNAs and differently regulate their fate in the cytoplasm of motoneuron-like cells, therefore suggesting complementary roles in neuronal RNA metabolism and neurodegeneration.

      Introduction

      The DNA/RNA-binding protein TDP-43 represents the major component of the intracellular inclusions occurring in the brain of patients affected by a series of neurodegenerative diseases, including the majority of both familial and sporadic amyotrophic lateral sclerosis (ALS)
      The abbreviations used are: ALS
      amyotrophic lateral sclerosis
      FTLD
      frontotemporal lobar dementia
      RBP
      RNA-binding protein
      RNP
      ribonucleoprotein
      IP
      immunoprecipitation
      RIP-chip
      RNA immunoprecipitation and chip analysis
      CLIP
      cross-linking and immunoprecipitation
      PAR-CLIP
      photoactivatable ribonucleoside-enhanced CLIP
      MEME
      multiple expectation maximization for motif elicitation
      RSAT
      regulatory sequence analysis tools
      PGRN
      progranulin
      OPTN
      optineurin.
      cases and a subset of Tau-negative and ubiquitin-positive frontotemporal lobar dementia (FTLD) cases (
      • Neumann M.
      • Sampathu D.M.
      • Kwong L.K.
      • Truax A.C.
      • Micsenyi M.C.
      • Chou T.T.
      • Bruce J.
      • Schuck T.
      • Grossman M.
      • Clark C.M.
      • McCluskey L.F.
      • Miller B.L.
      • Masliah E.
      • Mackenzie I.R.
      • Feldman H.
      • Feiden W.
      • Kretzschmar H.A.
      • Trojanowski J.Q.
      • Lee V.M.
      Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
      ,
      • Arai T.
      • Hasegawa M.
      • Akiyama H.
      • Ikeda K.
      • Nonaka T.
      • Mori H.
      • Mann D.
      • Tsuchiya K.
      • Yoshida M.
      • Hashizume Y.
      • Oda T.
      TDP-43 is a component of ubiquitin-positive Tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
      ). The genetic findings of causative mutations in TARDBP, the gene encoding for TDP-43, in 5% of familial ALS cases further support the pathogenic role of this protein (
      • Sreedharan J.
      • Blair I.P.
      • Tripathi V.B.
      • Hu X.
      • Vance C.
      • Rogelj B.
      • Ackerley S.
      • Durnall J.C.
      • Williams K.L.
      • Buratti E.
      • Baralle F.
      • de Belleroche J.
      • Mitchell J.D.
      • Leigh P.N.
      • Al-Chalabi A.
      • Miller C.C.
      • Nicholson G.
      • Shaw C.E.
      TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis.
      ). Abnormal cytoplasmic aggregates of FUS, another DNA/RNA-binding protein, are observed in a subset of FTLD cases, which are Tau- and TDP-43-negative, and in 4–5% of familial ALS cases with mutations in the FUS/TLS gene, suggesting that dysregulation of RNA metabolism plays an important role in ALS and FTLD pathogenesis (
      • Mackenzie I.R.
      • Rademakers R.
      • Neumann M.
      TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia.
      ,
      • Lagier-Tourenne C.
      • Polymenidou M.
      • Cleveland D.W.
      TDP-43 and FUS/TLS. Emerging roles in RNA processing and neurodegeneration.
      ).
      TDP-43 and FUS are ubiquitously expressed and multifunctional RNA-binding proteins (RBP) with a main localization in the nucleus, where they are implicated in several steps of RNA metabolism, such as transcription, pre-mRNA splicing, and microRNA processing (
      • van Blitterswijk M.
      • Landers J.E.
      RNA processing pathways in amyotrophic lateral sclerosis.
      ,
      • Kawahara Y.
      • Mieda-Sato A.
      TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes.
      ). However, they also take part in other cellular processes in the cytoplasmic compartment, including mRNA transport, mRNA stability, and translation (
      • Buratti E.
      • Baralle F.E.
      The multiple roles of TDP-43 in pre-mRNA processing and gene expression regulation.
      ,
      • Colombrita C.
      • Onesto E.
      • Tiloca C.
      • Ticozzi N.
      • Silani V.
      • Ratti A.
      RNA-binding proteins and RNA metabolism. A new scenario in the pathogenesis of amyotrophic lateral sclerosis.
      ). In fact, shuttling of these two proteins into the cytoplasm has been described, particularly in neurons, where an activity-dependent translocation of TDP-43 and FUS into dendrites was observed (
      • Wang I.F.
      • Wu L.S.
      • Chang H.Y.
      • Shen C.K.
      TDP-43, the signature protein of FTLD-U, is a neuronal activity-responsive factor.
      ,
      • Fujii R.
      • Okabe S.
      • Urushido T.
      • Inoue K.
      • Yoshimura A.
      • Tachibana T.
      • Nishikawa T.
      • Hicks G.G.
      • Takumi T.
      The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology.
      ,
      • Fujii R.
      • Takumi T.
      TLS facilitates transport of mRNA encoding an actin-stabilizing protein to dendritic spines.
      ,
      • Belly A.
      • Moreau-Gachelin F.
      • Sadoul R.
      • Goldberg Y.
      Delocalization of the multifunctional RNA splicing factor TLS/FUS in hippocampal neurones. Exclusion from the nucleus and accumulation in dendritic granules and spine heads.
      ). This suggests that these two RBPs participate also in regulating mRNA transport into neurites and, likely, local protein synthesis at synapses, two processes that are essential to neurons for a fast response to stimuli and cell survival (
      • Besse F.
      • Ephrussi A.
      Translational control of localized mRNAs. Restricting protein synthesis in space and time.
      ). Localization of TDP-43 and mutant FUS in stress granules, ribonucleoprotein (RNP) complexes that negatively control mRNA translation in condition of cellular insults, has been recently and widely demonstrated (
      • Colombrita C.
      • Zennaro E.
      • Fallini C.
      • Weber M.
      • Sommacal A.
      • Buratti E.
      • Silani V.
      • Ratti A.
      TDP-43 is recruited to stress granules in conditions of oxidative insult.
      ,
      • Bosco D.A.
      • Lemay N.
      • Ko H.K.
      • Zhou H.
      • Burke C.
      • Kwiatkowski Jr., T.J.
      • Sapp P.
      • McKenna-Yasek D.
      • Brown Jr., R.H.
      • Hayward L.J.
      Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules.
      ,
      • Gal J.
      • Zhang J.
      • Kwinter D.M.
      • Zhai J.
      • Jia H.
      • Jia J.
      • Zhu H.
      ), supporting an additional role of TDP-43 and FUS in the cytoplasmic compartment in association with translation.
      To date, however, disease mechanisms for these two RBPs have not been clearly elucidated. Mislocalization and aggregation of TDP-43 and FUS in the cytoplasm of ALS/FTLD-affected neurons are supposed to trigger neurodegeneration by loss of their biological functions (“loss-of-function” hypothesis) and/or by acquisition of potentially toxic functions in the cytoplasm (“gain-of-function” hypothesis) (
      • Da Cruz S.
      • Cleveland D.W.
      Understanding the role of TDP-43 and FUS/TLS in ALS and beyond.
      ). An important issue in understanding the pathogenic mechanisms in ALS and FTLD is certainly the full definition of the transcripts that are directly bound and post-transcriptionally regulated by TDP-43 and FUS in association with splicing and, in particular in neuronal cells, with mRNA transport, stabilization, and/or translational processes.
      Interestingly, using high throughput RNA/DNA sequencing technologies coupled to immunoprecipitation (RIP-seq) or cross-linking and immunoprecipitation (PAR-CLIP/iCLIP/CLIP-seq), recent papers have identified large sets of putative TDP-43 and FUS RNA targets in neuronal and non-neuronal cells (
      • Sephton C.F.
      • Cenik C.
      • Kucukural A.
      • Dammer E.B.
      • Cenik B.
      • Han Y.
      • Dewey C.M.
      • Roth F.P.
      • Herz J.
      • Peng J.
      • Moore M.J.
      • Yu G.
      Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes.
      ,
      • Tollervey J.R.
      • Curk T.
      • Rogelj B.
      • Briese M.
      • Cereda M.
      • Kayikci M.
      • König J.
      • Hortobágyi T.
      • Nishimura A.L.
      • Zupunski V.
      • Patani R.
      • Chandran S.
      • Rot G.
      • Zupan B.
      • Shaw C.E.
      • Ule J.
      Characterizing the RNA targets and position-dependent splicing regulation by TDP-43.
      ,
      • Polymenidou M.
      • Lagier-Tourenne C.
      • Hutt K.R.
      • Huelga S.C.
      • Moran J.
      • Liang T.Y.
      • Ling S.C.
      • Sun E.
      • Wancewicz E.
      • Mazur C.
      • Kordasiewicz H.
      • Sedaghat Y.
      • Donohue J.P.
      • Shiue L.
      • Bennett C.F.
      • Yeo G.W.
      • Cleveland D.W.
      Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43.
      ,
      • Xiao S.
      • Sanelli T.
      • Dib S.
      • Sheps D.
      • Findlater J.
      • Bilbao J.
      • Keith J.
      • Zinman L.
      • Rogaeva E.
      • Robertson J.
      RNA targets of TDP-43 identified by UV-CLIP are deregulated in ALS.
      ,
      • Hoell J.I.
      • Larsson E.
      • Runge S.
      • Nusbaum J.D.
      • Duggimpudi S.
      • Farazi T.A.
      • Hafner M.
      • Borkhardt A.
      • Sander C.
      • Tuschl T.
      RNA targets of wild-type and mutant FET family proteins.
      ). These studies revealed that TDP-43 and FUS are mainly involved in pre-mRNA splicing, as target sequences were preferentially localized in long intronic regions and near splice site acceptors, respectively. From these findings, however, it emerged that about 5–16% of all TDP-43 and FUS target sequences also mapped in exonic regions, with a high enrichment in 3′- untranslated region (UTR) sequences (3–12%) (
      • Sephton C.F.
      • Cenik C.
      • Kucukural A.
      • Dammer E.B.
      • Cenik B.
      • Han Y.
      • Dewey C.M.
      • Roth F.P.
      • Herz J.
      • Peng J.
      • Moore M.J.
      • Yu G.
      Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes.
      ,
      • Tollervey J.R.
      • Curk T.
      • Rogelj B.
      • Briese M.
      • Cereda M.
      • Kayikci M.
      • König J.
      • Hortobágyi T.
      • Nishimura A.L.
      • Zupunski V.
      • Patani R.
      • Chandran S.
      • Rot G.
      • Zupan B.
      • Shaw C.E.
      • Ule J.
      Characterizing the RNA targets and position-dependent splicing regulation by TDP-43.
      ,
      • Polymenidou M.
      • Lagier-Tourenne C.
      • Hutt K.R.
      • Huelga S.C.
      • Moran J.
      • Liang T.Y.
      • Ling S.C.
      • Sun E.
      • Wancewicz E.
      • Mazur C.
      • Kordasiewicz H.
      • Sedaghat Y.
      • Donohue J.P.
      • Shiue L.
      • Bennett C.F.
      • Yeo G.W.
      • Cleveland D.W.
      Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43.
      ,
      • Xiao S.
      • Sanelli T.
      • Dib S.
      • Sheps D.
      • Findlater J.
      • Bilbao J.
      • Keith J.
      • Zinman L.
      • Rogaeva E.
      • Robertson J.
      RNA targets of TDP-43 identified by UV-CLIP are deregulated in ALS.
      ,
      • Hoell J.I.
      • Larsson E.
      • Runge S.
      • Nusbaum J.D.
      • Duggimpudi S.
      • Farazi T.A.
      • Hafner M.
      • Borkhardt A.
      • Sander C.
      • Tuschl T.
      RNA targets of wild-type and mutant FET family proteins.
      ). In general, regulatory cis-acting elements, usually present in the 3′-UTR but sometimes also located in the 5′-UTR or in the coding sequence of target mRNAs, are responsible for RBP-mediated transport of mRNAs and post-transcriptional regulation of gene expression together with miRNA trans-acting factors (
      • Keene J.D.
      RNA regulons. Coordination of post-transcriptional events.
      ). The importance of TDP-43 and FUS binding to 3′-UTR regulatory sequences has been highlighted by the observation that TDP-43 can auto-regulate its own protein levels by binding to its 3′-UTR sequence in a negative feedback loop (
      • Ayala Y.M.
      • De Conti L.
      • Avendaño-Vázquez S.E.
      • Dhir A.
      • Romano M.
      • D'Ambrogio A.
      • Tollervey J.
      • Ule J.
      • Baralle M.
      • Buratti E.
      • Baralle F.E.
      TDP-43 regulates its mRNA levels through a negative feedback loop.
      ), whereas FUS was shown to transport the actin-related Nd1-L mRNA into dendrites by binding to its 3′-UTR (
      • Fujii R.
      • Takumi T.
      TLS facilitates transport of mRNA encoding an actin-stabilizing protein to dendritic spines.
      ).
      As TDP-43 and FUS share many common functional and biochemical features, we performed a RIP-chip analysis to identify and compare the biological mRNA targets of these two RBPs associated with RNP complexes in the cytoplasm of motoneuronal NSC-34 cells with the final aim of unraveling their potential role in mRNA transport, stability, and translation in neurons.

      DISCUSSION

      In this study, by utilizing a RIP-chip analysis, we defined and compared the mature mRNA targets or “targetome” of TDP-43 and FUS in the cytoplasmic compartment of motoneuron-like cells, which probably reflects a role of these two RBPs in RNA granule transport and local translation. Two distinct targetomes were identified for TDP-43 and FUS, and different post-transcriptional regulatory activities on their mRNA targets were demonstrated, suggesting that these two RBPs have distinct roles in neuronal RNA metabolism.
      Importantly, recent findings have contributed to better understanding the role of TDP-43 and FUS in the pathogenesis of ALS and FTLD. Disease models in yeast and Drosophila indicate that these two proteins may have distinct and complementary roles in triggering cell toxicity and neurodegeneration (
      • Da Cruz S.
      • Cleveland D.W.
      Understanding the role of TDP-43 and FUS/TLS in ALS and beyond.
      ,
      • Sun Z.
      • Diaz Z.
      • Fang X.
      • Hart M.P.
      • Chesi A.
      • Shorter J.
      • Gitler A.D.
      Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS.
      ,
      • Wang J.W.
      • Brent J.R.
      • Tomlinson A.
      • Shneider N.A.
      • McCabe B.D.
      The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span.
      ,
      • Lanson Jr., N.A.
      • Maltare A.
      • King H.
      • Smith R.
      • Kim J.H.
      • Taylor J.P.
      • Lloyd T.E.
      • Pandey U.B.
      A Drosophila model of FUS-related neurodegeneration reveals genetic interaction between FUS and TDP-43.
      ,
      • Kryndushkin D.
      • Wickner R.B.
      • Shewmaker F.
      FUS/TLS forms cytoplasmic aggregates, inhibits cell growth, and interacts with TDP-43 in a yeast model of amyotrophic lateral sclerosis.
      ), although the issue of whether they have common or distinct functions in the brain is still open. Certainly, the full definition of their biological role and, therefore, of their RNA targets in neuronal cells may help understand why their dysfunction, probably associated to their mislocalization and aggregation in the cytoplasm, leads to selective neuronal death in ALS and FTLD.
      The recent massive sequencing approaches utilized to identify the RNA targets of TDP-43 and FUS in brain tissues or in non-neural cells have confirmed and reinforced the role of these two RBPs mainly in pre-mRNA splicing activity. TDP-43 was found to recognize long intronic sequences endowed with (UG)n repeats (
      • Sephton C.F.
      • Cenik C.
      • Kucukural A.
      • Dammer E.B.
      • Cenik B.
      • Han Y.
      • Dewey C.M.
      • Roth F.P.
      • Herz J.
      • Peng J.
      • Moore M.J.
      • Yu G.
      Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes.
      ,
      • Tollervey J.R.
      • Curk T.
      • Rogelj B.
      • Briese M.
      • Cereda M.
      • Kayikci M.
      • König J.
      • Hortobágyi T.
      • Nishimura A.L.
      • Zupunski V.
      • Patani R.
      • Chandran S.
      • Rot G.
      • Zupan B.
      • Shaw C.E.
      • Ule J.
      Characterizing the RNA targets and position-dependent splicing regulation by TDP-43.
      ,
      • Polymenidou M.
      • Lagier-Tourenne C.
      • Hutt K.R.
      • Huelga S.C.
      • Moran J.
      • Liang T.Y.
      • Ling S.C.
      • Sun E.
      • Wancewicz E.
      • Mazur C.
      • Kordasiewicz H.
      • Sedaghat Y.
      • Donohue J.P.
      • Shiue L.
      • Bennett C.F.
      • Yeo G.W.
      • Cleveland D.W.
      Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43.
      ), whereas FUS preferentially bound near splice site acceptors (
      • Hoell J.I.
      • Larsson E.
      • Runge S.
      • Nusbaum J.D.
      • Duggimpudi S.
      • Farazi T.A.
      • Hafner M.
      • Borkhardt A.
      • Sander C.
      • Tuschl T.
      RNA targets of wild-type and mutant FET family proteins.
      ). However, the evidence that a smaller proportion of TDP-43 and FUS target sequences resides in the exonic and, more precisely, in the 3′-UTR sequences, also suggests that these two proteins may have additional roles in neuronal cells. In fact, similarly to the SMN protein, whose absence is causative of the infantile spinal muscular atrophy (SMA), TDP-43, and FUS RBPs, may have not only a nuclear activity associated to pre-mRNA splicing but also a role in mRNA transport and local translation in neurites, which may better account for neuronal cell death in ALS and FTLD diseases.
      Several experimental findings already suggest that these two RBPs participate to transport of RNA granules in neurons and/or may control mRNA stability and translation. TDP-43 was described to bind and transport β-actin and calmodulin kinase II mRNAs to dendrites upon depolarization of hippocampal primary neurons and to regulate the stability of Nfl and its own transcript by binding to their 3′-UTR sequences (
      • Wang I.F.
      • Wu L.S.
      • Chang H.Y.
      • Shen C.K.
      TDP-43, the signature protein of FTLD-U, is a neuronal activity-responsive factor.
      ,
      • Ayala Y.M.
      • De Conti L.
      • Avendaño-Vázquez S.E.
      • Dhir A.
      • Romano M.
      • D'Ambrogio A.
      • Tollervey J.
      • Ule J.
      • Baralle M.
      • Buratti E.
      • Baralle F.E.
      TDP-43 regulates its mRNA levels through a negative feedback loop.
      ,
      • Strong M.J.
      • Volkening K.
      • Hammond R.
      • Yang W.
      • Strong W.
      • Leystra-Lantz C.
      • Shoesmith C.
      TDP43 is a human low molecular weight neurofilament (hNFL) mRNA-binding protein.
      ). Similarly, FUS was shown to bind Nd1-L mRNA in its 3′-UTR and to move into dendrites upon NMDA stimulation (
      • Fujii R.
      • Okabe S.
      • Urushido T.
      • Inoue K.
      • Yoshimura A.
      • Tachibana T.
      • Nishikawa T.
      • Hicks G.G.
      • Takumi T.
      The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology.
      ,
      • Fujii R.
      • Takumi T.
      TLS facilitates transport of mRNA encoding an actin-stabilizing protein to dendritic spines.
      ). The involvement of TDP-43 in controlling mRNA translation is based on the indirect evidence that it can be part of stress granules (
      • Colombrita C.
      • Zennaro E.
      • Fallini C.
      • Weber M.
      • Sommacal A.
      • Buratti E.
      • Silani V.
      • Ratti A.
      TDP-43 is recruited to stress granules in conditions of oxidative insult.
      ), the RNA triage site forming upon translational arrest induced by different cell stressors and containing stalled ribosomes, translation factors, and mRNAs as well as several RBPs (
      • Anderson P.
      • Kedersha N.
      Stress granules. The Tao of RNA triage.
      ). On the other hand, FUS forms stress granules only when it is mutated (
      • Bosco D.A.
      • Lemay N.
      • Ko H.K.
      • Zhou H.
      • Burke C.
      • Kwiatkowski Jr., T.J.
      • Sapp P.
      • McKenna-Yasek D.
      • Brown Jr., R.H.
      • Hayward L.J.
      Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules.
      ,
      • Gal J.
      • Zhang J.
      • Kwinter D.M.
      • Zhai J.
      • Jia H.
      • Jia J.
      • Zhu H.
      ,
      • Dormann D.
      • Rodde R.
      • Edbauer D.
      • Bentmann E.
      • Fischer I.
      • Hruscha A.
      • Than M.E.
      • Mackenzie I.R.
      • Capell A.
      • Schmid B.
      • Neumann M.
      • Haass C.
      ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import.
      ), and this supports the recent evidence that mutant FUS proteins, upon translocation into the cytoplasm, changes their targetome and preferentially binds 3′-UTR sequences (
      • Hoell J.I.
      • Larsson E.
      • Runge S.
      • Nusbaum J.D.
      • Duggimpudi S.
      • Farazi T.A.
      • Hafner M.
      • Borkhardt A.
      • Sander C.
      • Tuschl T.
      RNA targets of wild-type and mutant FET family proteins.
      ).
      TDP-43 has been described to co-localize with FUS mainly in nuclear complexes, and this interaction, which seems to be restricted to only 10% of cells, is greatly enhanced by mutant TDP-43 (
      • Kim S.H.
      • Shanware N.P.
      • Bowler M.J.
      • Tibbetts R.S.
      Amyotrophic lateral sclerosis-associated proteins TDP-43 and FUS/TLS function in a common biochemical complex to co-regulate HDAC6 mRNA.
      ,
      • Ling S.C.
      • Albuquerque C.P.
      • Han J.S.
      • Lagier-Tourenne C.
      • Tokunaga S.
      • Zhou H.
      • Cleveland D.W.
      ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS.
      ). TDP-43 and FUS were reported to co-localize also in cytoplasmic RNPs (
      • Kim S.H.
      • Shanware N.P.
      • Bowler M.J.
      • Tibbetts R.S.
      Amyotrophic lateral sclerosis-associated proteins TDP-43 and FUS/TLS function in a common biochemical complex to co-regulate HDAC6 mRNA.
      ), although the association of these proteins in the same RNP particle was rarely observed in our assays. Moreover, our RIP-chip data indicate that in the cytoplasm, TDP-43 and FUS recognize and bind distinct sets of mRNAs, suggesting that these two RBPs take part in distinct RNP complexes. Interestingly, we found that TDP-43 targets are enriched in transcripts associated to neuron-specific activities, whereas FUS targets are related to more general cellular activities, including DNA repair, cell cycle, and RNA processing, which have already been associated to FUS (
      • Wang X.
      • Arai S.
      • Song X.
      • Reichart D.
      • Du K.
      • Pascual G.
      • Tempst P.
      • Rosenfeld M.G.
      • Glass C.K.
      • Kurokawa R.
      Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription.
      ,
      • Kovar H.
      Dr. Jekyll and Mr. Hyde. The two faces of the FUS/EWS/TAF15 protein family.
      ). Although no common transcripts were identified from our analyses, the fact that some targets belonged to common GO categories indicates that TDP-43- and FUS-mediated post-transcriptional regulation may converge on the same cellular pathways.
      In general, bioinformatics analyses of our data set revealed that the well known (UG)n consensus binding sequence for TDP-43 was present in the 3′-UTR of our RIP-chip-identified targets in about 80% of cases. However, other exonic regions may be responsible for TDP-43 binding, as already shown for Hdac6 mRNA (
      • Fiesel F.C.
      • Voigt A.
      • Weber S.S.
      • Van den Haute C.
      • Waldenmaier A.
      • Görner K.
      • Walter M.
      • Anderson M.L.
      • Kern J.V.
      • Rasse T.M.
      • Schmidt T.
      • Springer W.
      • Kirchner R.
      • Bonin M.
      • Neumann M.
      • Baekelandt V.
      • Alunni-Fabbroni M.
      • Schulz J.B.
      • Kahle P.J.
      Knockdown of transactive response DNA-binding protein (TDP-43) down-regulates histone deacetylase 6.
      ), and other consensus binding motifs may be present in such 3′-UTR sequences, as also supported by our computational analyses. The issue is more complicated for FUS because the MEME-identified binding motif was represented only in a very small subset of 3′-UTR target sequences, and the short GGUG consensus motif, previously identified by a SELEX analysis (
      • Lerga A.
      • Hallier M.
      • Delva L.
      • Orvain C.
      • Gallais I.
      • Marie J.
      • Moreau-Gachelin F.
      Identification of an RNA binding specificity for the potential splicing factor TLS.
      ), was not consistently observed in our data set. It is also likely that FUS binding needs a particular folding and conformational structure of the target mRNA rather than a mere consensus sequence motif, as already suggested (
      • Fujii R.
      • Takumi T.
      TLS facilitates transport of mRNA encoding an actin-stabilizing protein to dendritic spines.
      ,
      • Hoell J.I.
      • Larsson E.
      • Runge S.
      • Nusbaum J.D.
      • Duggimpudi S.
      • Farazi T.A.
      • Hafner M.
      • Borkhardt A.
      • Sander C.
      • Tuschl T.
      RNA targets of wild-type and mutant FET family proteins.
      ). Nonetheless, by in vitro binding assays we have confirmed binding of FUS to the 3′-UTR of RIP-chip-identified targets in 3/5 selected cases, leaving again open the possibility of other exonic sequences being recognized and bound by FUS in these transcripts.
      The RIP-chip analysis was suitable to our purpose to define the mRNA composition of TDP-43- and FUS-containing RNPs in the cytoplasmic compartment of NSC-34 cells. We confirmed some TDP-43 and FUS targets recently identified by other approaches and roughly mapped in exonic regions (
      • Sephton C.F.
      • Cenik C.
      • Kucukural A.
      • Dammer E.B.
      • Cenik B.
      • Han Y.
      • Dewey C.M.
      • Roth F.P.
      • Herz J.
      • Peng J.
      • Moore M.J.
      • Yu G.
      Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes.
      ,
      • Tollervey J.R.
      • Curk T.
      • Rogelj B.
      • Briese M.
      • Cereda M.
      • Kayikci M.
      • König J.
      • Hortobágyi T.
      • Nishimura A.L.
      • Zupunski V.
      • Patani R.
      • Chandran S.
      • Rot G.
      • Zupan B.
      • Shaw C.E.
      • Ule J.
      Characterizing the RNA targets and position-dependent splicing regulation by TDP-43.
      ,
      • Polymenidou M.
      • Lagier-Tourenne C.
      • Hutt K.R.
      • Huelga S.C.
      • Moran J.
      • Liang T.Y.
      • Ling S.C.
      • Sun E.
      • Wancewicz E.
      • Mazur C.
      • Kordasiewicz H.
      • Sedaghat Y.
      • Donohue J.P.
      • Shiue L.
      • Bennett C.F.
      • Yeo G.W.
      • Cleveland D.W.
      Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43.
      ,
      • Hoell J.I.
      • Larsson E.
      • Runge S.
      • Nusbaum J.D.
      • Duggimpudi S.
      • Farazi T.A.
      • Hafner M.
      • Borkhardt A.
      • Sander C.
      • Tuschl T.
      RNA targets of wild-type and mutant FET family proteins.
      ), further suggesting that TDP-43 and FUS bind them as mature mRNAs. However, the targetomes we defined for TDP-43 and FUS may represent a subgroup of all their real targets. In fact, the discrepancy observed in the number of targets identified for TDP-43 and FUS might not reflect a difference in their RNA binding properties but more probably the different efficiency of the commercial antibodies used to recognize and immunoprecipitate these two proteins when they are complexed into RNP particles, as also observed in CLIP experiments (
      • Polymenidou M.
      • Lagier-Tourenne C.
      • Hutt K.R.
      • Huelga S.C.
      • Moran J.
      • Liang T.Y.
      • Ling S.C.
      • Sun E.
      • Wancewicz E.
      • Mazur C.
      • Kordasiewicz H.
      • Sedaghat Y.
      • Donohue J.P.
      • Shiue L.
      • Bennett C.F.
      • Yeo G.W.
      • Cleveland D.W.
      Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43.
      ,
      • Xiao S.
      • Sanelli T.
      • Dib S.
      • Sheps D.
      • Findlater J.
      • Bilbao J.
      • Keith J.
      • Zinman L.
      • Rogaeva E.
      • Robertson J.
      RNA targets of TDP-43 identified by UV-CLIP are deregulated in ALS.
      ). This could also explain the reason why we did not identify some recently demonstrated TDP-43 and FUS targets as mature mRNAs (
      • Wang I.F.
      • Wu L.S.
      • Chang H.Y.
      • Shen C.K.
      TDP-43, the signature protein of FTLD-U, is a neuronal activity-responsive factor.
      ,
      • Ayala Y.M.
      • De Conti L.
      • Avendaño-Vázquez S.E.
      • Dhir A.
      • Romano M.
      • D'Ambrogio A.
      • Tollervey J.
      • Ule J.
      • Baralle M.
      • Buratti E.
      • Baralle F.E.
      TDP-43 regulates its mRNA levels through a negative feedback loop.
      ,
      • Strong M.J.
      • Volkening K.
      • Hammond R.
      • Yang W.
      • Strong W.
      • Leystra-Lantz C.
      • Shoesmith C.
      TDP43 is a human low molecular weight neurofilament (hNFL) mRNA-binding protein.
      ,
      • Fiesel F.C.
      • Voigt A.
      • Weber S.S.
      • Van den Haute C.
      • Waldenmaier A.
      • Görner K.
      • Walter M.
      • Anderson M.L.
      • Kern J.V.
      • Rasse T.M.
      • Schmidt T.
      • Springer W.
      • Kirchner R.
      • Bonin M.
      • Neumann M.
      • Baekelandt V.
      • Alunni-Fabbroni M.
      • Schulz J.B.
      • Kahle P.J.
      Knockdown of transactive response DNA-binding protein (TDP-43) down-regulates histone deacetylase 6.
      ). Notwithstanding these caveats, the data emerging from our work undoubtedly contribute to better define the cytoplasmic targetome of TDP-43 and FUS, a result that will certainly represent an important referential source to begin to unravel the pathogenic mechanisms involved in ALS and FTLD diseases. Nonetheless, as shown by our in vitro analyses, all these “-omics” data need to be carefully handled and to be experimentally validated in biological models.
      In keeping with this consideration, in this work we have further investigated the role played by TDP-43 in regulating post-transcriptionally Vegfa and Grn, as they are genetically related to ALS and FTLD (
      • Lambrechts D.
      • Storkebaum E.
      • Morimoto M.
      • Del-Favero J.
      • Desmet F.
      • Marklund S.L.
      • Wyns S.
      • Thijs V.
      • Andersson J.
      • van Marion I.
      • Al-Chalabi A.
      • Bornes S.
      • Musson R.
      • Hansen V.
      • Beckman L.
      • Adolfsson R.
      • Pall H.S.
      • Prats H.
      • Vermeire S.
      • Rutgeerts P.
      • Katayama S.
      • Awata T.
      • Leigh N.
      • Lang-Lazdunski L.
      • Dewerchin M.
      • Shaw C.
      • Moons L.
      • Vlietinck R.
      • Morrison K.E.
      • Robberecht W.
      • Van Broeckhoven C.
      • Collen D.
      • Andersen P.M.
      • Carmeliet P.
      VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death.
      ,
      • Baker M.
      • Mackenzie I.R.
      • Pickering-Brown S.M.
      • Gass J.
      • Rademakers R.
      • Lindholm C.
      • Snowden J.
      • Adamson J.
      • Sadovnick A.D.
      • Rollinson S.
      • Cannon A.
      • Dwosh E.
      • Neary D.
      • Melquist S.
      • Richardson A.
      • Dickson D.
      • Berger Z.
      • Eriksen J.
      • Robinson T.
      • Zehr C.
      • Dickey C.A.
      • Crook R.
      • McGowan E.
      • Mann D.
      • Boeve B.
      • Feldman H.
      • Hutton M.
      Mutations in progranulin cause Tau-negative frontotemporal dementia linked to chromosome 17.
      ,
      • Cruts M.
      • Gijselinck I.
      • van der Zee J.
      • Engelborghs S.
      • Wils H.
      • Pirici D.
      • Rademakers R.
      • Vandenberghe R.
      • Dermaut B.
      • Martin J.J.
      • van Duijn C.
      • Peeters K.
      • Sciot R.
      • Santens P.
      • De Pooter T.
      • Mattheijssens M.
      • Van den Broeck M.
      • Cuijt I.
      • Vennekens K.
      • De Deyn P.P.
      • Kumar-Singh S.
      • Van Broeckhoven C.
      Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.
      ,
      • Borroni B.
      • Ghezzi S.
      • Agosti C.
      • Archetti S.
      • Fenoglio C.
      • Galimberti D.
      • Scarpini E.
      • Di Luca M.
      • Bresolin N.
      • Comi G.P.
      • Padovani A.
      • Del Bo R.
      Preliminary evidence that VEGF genetic variability confers susceptibility to frontotemporal lobar degeneration.
      ,
      • Sleegers K.
      • Brouwers N.
      • Maurer-Stroh S.
      • van Es M.A.
      • Van Damme P.
      • van Vught P.W.
      • van der Zee J.
      • Serneels S.
      • De Pooter T.
      • Van den Broeck M.
      • Cruts M.
      • Schymkowitz J.
      • De Jonghe P.
      • Rousseau F.
      • van den Berg L.H.
      • Robberecht W.
      • Van Broeckhoven C.
      Progranulin genetic variability contributes to amyotrophic lateral sclerosis.
      ) and their reduced protein levels have been shown to trigger neuronal death (
      • Van Damme P.
      • Van Hoecke A.
      • Lambrechts D.
      • Vanacker P.
      • Bogaert E.
      • van Swieten J.
      • Carmeliet P.
      • Van Den Bosch L.
      • Robberecht W.
      Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival.
      ,
      • Sathasivam S.
      VEGF and ALS.
      ). We found that TDP-43, by binding to Vegfa and Grn 3′-UTR sequences, may control their mRNA stability and ultimately determine the content of PGRN protein. Also, the most common TDP-43 mutations reported in ALS patients (Q331K, M337V, A382T) had no effect on its binding activity of Vegfa and Grn target mRNAs.
      Conversely, FUS binding to Taf15, Nvl, and Vps54 3′-UTRs seems to regulate neither the stability of its bound targets nor the content of VPS54 protein, for which an immunoblot was possible. Interestingly, this observation about FUS was already reported for the Nd1-L target, which was described to be transported into dendrites by FUS with no regulation of its mRNA stability (
      • Fujii R.
      • Takumi T.
      TLS facilitates transport of mRNA encoding an actin-stabilizing protein to dendritic spines.
      ).
      Importantly, an increase in PGRN protein levels and no changes in VEGF content were also detected in post-mortem spinal cord tissues from ALS patients (
      • Irwin D.
      • Lippa C.F.
      • Rosso A.
      Progranulin (PGRN) expression in ALS. An immunohistochemical study.
      ,
      • Devos D.
      • Moreau C.
      • Lassalle P.
      • Perez T.
      • De Seze J.
      • Brunaud-Danel V.
      • Destée A.
      • Tonnel A.B.
      • Just N.
      Low levels of the vascular endothelial growth factor in CSF from early ALS patients.
      ), similarly to what we observed in our in vitro model of TDP-43 “loss of function” condition. Indeed, in “TDP-43 proteinopathy” the nuclear clearance of TDP-43 together with its sequestration into aggregates may determine reduced levels of its soluble and active form (
      • Dormann D.
      • Haass C.
      TDP-43 and FUS: a nuclear affair.
      ). Therefore, our experimental data provide a mechanistic explanation of what is observed in affected tissues of sporadic ALS and FTLD patients. Moreover, our results indicate that changes of TDP-43 levels may in turn change PGRN content, which needs to be strictly regulated to avoid neurodegeneration or cancer (
      • Gass J.
      • Prudencio M.
      • Stetler C.
      • Petrucelli L.
      Progranulin: An emerging target for FTLD therapies.
      ). We also postulate that the observed unchanged levels of endogenous VEGF upon TDP-43 depletion or overexpression may be determined by the delicate balance of different trans-acting factors, including miRNAs and the stabilizing ELAV RBPs (
      • Levy N.S.
      • Chung S.
      • Furneaux H.
      • Levy A.P.
      Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR.
      ), which exert a complex post-transcriptional regulation on the long Vegfa 3′-UTR.
      In conclusion, the findings emerging from our work further support the idea that TDP-43 and FUS have different, but probably complementary, functions in the cytoplasmic compartment of neuronal cells, such as controlling mRNA transport, stability, and probably translation in the case of TDP-43 and mRNA transport into neuronal processes in the case of FUS. Post-transcriptional regulation of gene expression is a particularly complex and articulated mechanism, above all in highly specialized cells such as neurons, where an efficient and tight control of mRNA fate in the nucleus and in the cytoplasm together with its transport into neurites for local translation is important for their demanding metabolism. But most importantly, our study further highlights the potential importance of aberrant RNA metabolism as a direct cause of disease. In fact, considering the sheer number of potentially mysregulated targets/pathways after changes in cellular distribution of TDP-43 and FUS protein, it is very likely that even small alterations in the relative quantity of these proteins in the nucleus/cytoplasm might cause neurodegeneration over a long period of time (especially if we consider that the ability of cells to compensate for even small changes from normality gradually diminishes with age). One of the key research questions that studies such as ours will open up in the near future will be to correctly categorize and classify these aberrant events in terms of severity/importance with regard to the neurodegeneration process. This will in turn help to prioritize areas of therapeutic intervention.

      Acknowledgments

      We thank Dr. D. Gentilini for support in chip hybridization and data analysis.

      REFERENCES

        • Neumann M.
        • Sampathu D.M.
        • Kwong L.K.
        • Truax A.C.
        • Micsenyi M.C.
        • Chou T.T.
        • Bruce J.
        • Schuck T.
        • Grossman M.
        • Clark C.M.
        • McCluskey L.F.
        • Miller B.L.
        • Masliah E.
        • Mackenzie I.R.
        • Feldman H.
        • Feiden W.
        • Kretzschmar H.A.
        • Trojanowski J.Q.
        • Lee V.M.
        Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
        Science. 2006; 314: 130-133
        • Arai T.
        • Hasegawa M.
        • Akiyama H.
        • Ikeda K.
        • Nonaka T.
        • Mori H.
        • Mann D.
        • Tsuchiya K.
        • Yoshida M.
        • Hashizume Y.
        • Oda T.
        TDP-43 is a component of ubiquitin-positive Tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
        Biochem. Biophys. Res. Commun. 2006; 351: 602-611
        • Sreedharan J.
        • Blair I.P.
        • Tripathi V.B.
        • Hu X.
        • Vance C.
        • Rogelj B.
        • Ackerley S.
        • Durnall J.C.
        • Williams K.L.
        • Buratti E.
        • Baralle F.
        • de Belleroche J.
        • Mitchell J.D.
        • Leigh P.N.
        • Al-Chalabi A.
        • Miller C.C.
        • Nicholson G.
        • Shaw C.E.
        TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis.
        Science. 2008; 319: 1668-1672
        • Mackenzie I.R.
        • Rademakers R.
        • Neumann M.
        TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia.
        Lancet Neurol. 2010; 9: 995-1007
        • Lagier-Tourenne C.
        • Polymenidou M.
        • Cleveland D.W.
        TDP-43 and FUS/TLS. Emerging roles in RNA processing and neurodegeneration.
        Hum. Mol. Genet. 2010; 19: R46-R64
        • van Blitterswijk M.
        • Landers J.E.
        RNA processing pathways in amyotrophic lateral sclerosis.
        Neurogenetics. 2010; 11: 275-290
        • Kawahara Y.
        • Mieda-Sato A.
        TDP-43 promotes microRNA biogenesis as a component of the Drosha and Dicer complexes.
        Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 3347-3352
        • Buratti E.
        • Baralle F.E.
        The multiple roles of TDP-43 in pre-mRNA processing and gene expression regulation.
        RNA Biol. 2010; 7: 420-429
        • Colombrita C.
        • Onesto E.
        • Tiloca C.
        • Ticozzi N.
        • Silani V.
        • Ratti A.
        RNA-binding proteins and RNA metabolism. A new scenario in the pathogenesis of amyotrophic lateral sclerosis.
        Arch. Ital. Biol. 2011; 149: 83-99
        • Wang I.F.
        • Wu L.S.
        • Chang H.Y.
        • Shen C.K.
        TDP-43, the signature protein of FTLD-U, is a neuronal activity-responsive factor.
        J. Neurochem. 2008; 105: 797-806
        • Fujii R.
        • Okabe S.
        • Urushido T.
        • Inoue K.
        • Yoshimura A.
        • Tachibana T.
        • Nishikawa T.
        • Hicks G.G.
        • Takumi T.
        The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology.
        Curr. Biol. 2005; 15: 587-593
        • Fujii R.
        • Takumi T.
        TLS facilitates transport of mRNA encoding an actin-stabilizing protein to dendritic spines.
        J. Cell Sci. 2005; 118: 5755-5765
        • Belly A.
        • Moreau-Gachelin F.
        • Sadoul R.
        • Goldberg Y.
        Delocalization of the multifunctional RNA splicing factor TLS/FUS in hippocampal neurones. Exclusion from the nucleus and accumulation in dendritic granules and spine heads.
        Neurosci. Lett. 2005; 379: 152-157
        • Besse F.
        • Ephrussi A.
        Translational control of localized mRNAs. Restricting protein synthesis in space and time.
        Nat. Rev. Mol. Cell Biol. 2008; 9: 971-980
        • Colombrita C.
        • Zennaro E.
        • Fallini C.
        • Weber M.
        • Sommacal A.
        • Buratti E.
        • Silani V.
        • Ratti A.
        TDP-43 is recruited to stress granules in conditions of oxidative insult.
        J. Neurochem. 2009; 111: 1051-1061
        • Bosco D.A.
        • Lemay N.
        • Ko H.K.
        • Zhou H.
        • Burke C.
        • Kwiatkowski Jr., T.J.
        • Sapp P.
        • McKenna-Yasek D.
        • Brown Jr., R.H.
        • Hayward L.J.
        Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules.
        Hum. Mol. Genet. 2010; 19: 4160-4175
        • Gal J.
        • Zhang J.
        • Kwinter D.M.
        • Zhai J.
        • Jia H.
        • Jia J.
        • Zhu H.
        Neurobiol. Aging. 2011; 32: 2323-2340
        • Da Cruz S.
        • Cleveland D.W.
        Understanding the role of TDP-43 and FUS/TLS in ALS and beyond.
        Curr. Opin. Neurobiol. 2011; 21: 904-919
        • Sephton C.F.
        • Cenik C.
        • Kucukural A.
        • Dammer E.B.
        • Cenik B.
        • Han Y.
        • Dewey C.M.
        • Roth F.P.
        • Herz J.
        • Peng J.
        • Moore M.J.
        • Yu G.
        Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes.
        J. Biol. Chem. 2011; 286: 1204-1215
        • Tollervey J.R.
        • Curk T.
        • Rogelj B.
        • Briese M.
        • Cereda M.
        • Kayikci M.
        • König J.
        • Hortobágyi T.
        • Nishimura A.L.
        • Zupunski V.
        • Patani R.
        • Chandran S.
        • Rot G.
        • Zupan B.
        • Shaw C.E.
        • Ule J.
        Characterizing the RNA targets and position-dependent splicing regulation by TDP-43.
        Nat. Neurosci. 2011; 14: 452-458
        • Polymenidou M.
        • Lagier-Tourenne C.
        • Hutt K.R.
        • Huelga S.C.
        • Moran J.
        • Liang T.Y.
        • Ling S.C.
        • Sun E.
        • Wancewicz E.
        • Mazur C.
        • Kordasiewicz H.
        • Sedaghat Y.
        • Donohue J.P.
        • Shiue L.
        • Bennett C.F.
        • Yeo G.W.
        • Cleveland D.W.
        Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43.
        Nat. Neurosci. 2011; 14: 459-468
        • Xiao S.
        • Sanelli T.
        • Dib S.
        • Sheps D.
        • Findlater J.
        • Bilbao J.
        • Keith J.
        • Zinman L.
        • Rogaeva E.
        • Robertson J.
        RNA targets of TDP-43 identified by UV-CLIP are deregulated in ALS.
        Mol. Cell. Neurosci. 2011; 47: 167-180
        • Hoell J.I.
        • Larsson E.
        • Runge S.
        • Nusbaum J.D.
        • Duggimpudi S.
        • Farazi T.A.
        • Hafner M.
        • Borkhardt A.
        • Sander C.
        • Tuschl T.
        RNA targets of wild-type and mutant FET family proteins.
        Nat. Struct. Mol. Biol. 2011; 18: 1428-1431
        • Keene J.D.
        RNA regulons. Coordination of post-transcriptional events.
        Nat. Rev. Genet. 2007; 8: 533-543
        • Ayala Y.M.
        • De Conti L.
        • Avendaño-Vázquez S.E.
        • Dhir A.
        • Romano M.
        • D'Ambrogio A.
        • Tollervey J.
        • Ule J.
        • Baralle M.
        • Buratti E.
        • Baralle F.E.
        TDP-43 regulates its mRNA levels through a negative feedback loop.
        EMBO J. 2011; 30: 277-288
        • Ratti A.
        • Fallini C.
        • Colombrita C.
        • Pascale A.
        • Laforenza U.
        • Quattrone A.
        • Silani V.
        Post-transcriptional regulation of neuro-oncological ventral antigen 1 by the neuronal RNA-binding proteins ELAV.
        J. Biol. Chem. 2008; 283: 7531-7541
        • Machanick P.
        • Bailey T.L.
        MEME-ChIP. Motif analysis of large DNA datasets.
        Bioinformatics. 2011; 27: 1696-1697
        • Bailey T.L.
        • Williams N.
        • Misleh C.
        • Li W.W.
        MEME. Discovering and analyzing DNA and protein sequence motifs.
        Nucleic Acids Res. 2006; 34: W369-W373
        • Ayala Y.M.
        • Zago P.
        • D'Ambrogio A.
        • Xu Y.F.
        • Petrucelli L.
        • Buratti E.
        • Baralle F.E.
        Structural determinants of the cellular localization and shuttling of TDP-43.
        J. Cell Sci. 2008; 121: 3778-3785
        • Buratti E.
        • Baralle F.E.
        Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9.
        J. Biol. Chem. 2001; 276: 36337-36343
        • Ayala Y.M.
        • Pantano S.
        • D'Ambrogio A.
        • Buratti E.
        • Brindisi A.
        • Marchetti C.
        • Romano M.
        • Baralle F.E.
        Human, Drosophila, and C. elegans TDP43. Nucleic acid binding properties and splicing regulatory function.
        J. Mol. Biol. 2005; 348: 575-588
        • Lerga A.
        • Hallier M.
        • Delva L.
        • Orvain C.
        • Gallais I.
        • Marie J.
        • Moreau-Gachelin F.
        Identification of an RNA binding specificity for the potential splicing factor TLS.
        J. Biol. Chem. 2001; 276: 6807-6816
        • Lambrechts D.
        • Storkebaum E.
        • Morimoto M.
        • Del-Favero J.
        • Desmet F.
        • Marklund S.L.
        • Wyns S.
        • Thijs V.
        • Andersson J.
        • van Marion I.
        • Al-Chalabi A.
        • Bornes S.
        • Musson R.
        • Hansen V.
        • Beckman L.
        • Adolfsson R.
        • Pall H.S.
        • Prats H.
        • Vermeire S.
        • Rutgeerts P.
        • Katayama S.
        • Awata T.
        • Leigh N.
        • Lang-Lazdunski L.
        • Dewerchin M.
        • Shaw C.
        • Moons L.
        • Vlietinck R.
        • Morrison K.E.
        • Robberecht W.
        • Van Broeckhoven C.
        • Collen D.
        • Andersen P.M.
        • Carmeliet P.
        VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death.
        Nat. Genet. 2003; 34: 383-394
        • Baker M.
        • Mackenzie I.R.
        • Pickering-Brown S.M.
        • Gass J.
        • Rademakers R.
        • Lindholm C.
        • Snowden J.
        • Adamson J.
        • Sadovnick A.D.
        • Rollinson S.
        • Cannon A.
        • Dwosh E.
        • Neary D.
        • Melquist S.
        • Richardson A.
        • Dickson D.
        • Berger Z.
        • Eriksen J.
        • Robinson T.
        • Zehr C.
        • Dickey C.A.
        • Crook R.
        • McGowan E.
        • Mann D.
        • Boeve B.
        • Feldman H.
        • Hutton M.
        Mutations in progranulin cause Tau-negative frontotemporal dementia linked to chromosome 17.
        Nature. 2006; 442: 916-919
        • Cruts M.
        • Gijselinck I.
        • van der Zee J.
        • Engelborghs S.
        • Wils H.
        • Pirici D.
        • Rademakers R.
        • Vandenberghe R.
        • Dermaut B.
        • Martin J.J.
        • van Duijn C.
        • Peeters K.
        • Sciot R.
        • Santens P.
        • De Pooter T.
        • Mattheijssens M.
        • Van den Broeck M.
        • Cuijt I.
        • Vennekens K.
        • De Deyn P.P.
        • Kumar-Singh S.
        • Van Broeckhoven C.
        Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.
        Nature. 2006; 442: 920-924
        • Elden A.C.
        • Kim H.J.
        • Hart M.P.
        • Chen-Plotkin A.S.
        • Johnson B.S.
        • Fang X.
        • Armakola M.
        • Geser F.
        • Greene R.
        • Lu M.M.
        • Padmanabhan A.
        • Clay-Falcone D.
        • McCluskey L.
        • Elman L.
        • Juhr D.
        • Gruber P.J.
        • Rüb U.
        • Auburger G.
        • Trojanowski J.Q.
        • Lee V.M.
        • Van Deerlin V.M.
        • Bonini N.M.
        • Gitler A.D.
        Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS.
        Nature. 2010; 466: 1069-1075
        • Maruyama H.
        • Morino H.
        • Ito H.
        • Izumi Y.
        • Kato H.
        • Watanabe Y.
        • Kinoshita Y.
        • Kamada M.
        • Nodera H.
        • Suzuki H.
        • Komure O.
        • Matsuura S.
        • Kobatake K.
        • Morimoto N.
        • Abe K.
        • Suzuki N.
        • Aoki M.
        • Kawata A.
        • Hirai T.
        • Kato T.
        • Ogasawara K.
        • Hirano A.
        • Takumi T.
        • Kusaka H.
        • Hagiwara K.
        • Kaji R.
        • Kawakami H.
        Mutations of optineurin in amyotrophic lateral sclerosis.
        Nature. 2010; 465: 223-226
        • Ticozzi N.
        • Vance C.
        • Leclerc A.L.
        • Keagle P.
        • Glass J.D.
        • McKenna-Yasek D.
        • Sapp P.C.
        • Silani V.
        • Bosco D.A.
        • Shaw C.E.
        • Brown Jr., R.H.
        • Landers J.E.
        Mutational analysis reveals the FUS homolog TAF15 as a candidate gene for familial amyotrophic lateral sclerosis.
        Am. J. Med. Genet. B. Neuropsychiatr. Genet. 2011; 156B: 285-290
        • Schmitt-John T.
        • Drepper C.
        • Mussmann A.
        • Hahn P.
        • Kuhlmann M.
        • Thiel C.
        • Hafner M.
        • Lengeling A.
        • Heimann P.
        • Jones J.M.
        • Meisler M.H.
        • Jockusch H.
        Mutation of Vps54 causes motor neuron disease and defective spermiogenesis in the wobbler mouse.
        Nat. Genet. 2005; 37: 1213-1215
        • Sharma A.
        • Lambrechts A.
        • Hao le T.
        • Le T.T.
        • Sewry C.A.
        • Ampe C.
        • Burghes A.H.
        • Morris G.E.
        A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells.
        Exp. Cell Res. 2005; 309: 185-197
        • Gorba T.
        • Bradoo P.
        • Antonic A.
        • Marvin K.
        • Liu D.X.
        • Lobie P.E.
        • Reymann K.G.
        • Gluckman P.D.
        • Sieg F.
        Neural regeneration protein is a novel chemoattractive and neuronal survival-promoting factor.
        Exp. Cell Res. 2006; 312: 3060-3074
        • Nagahama M.
        • Hara Y.
        • Seki A.
        • Yamazoe T.
        • Kawate Y.
        • Shinohara T.
        • Hatsuzawa K.
        • Tani K.
        • Tagaya M.
        NVL2 is a nucleolar AAA-ATPase that interacts with ribosomal protein L5 through its nucleolar localization sequence.
        Mol. Biol. Cell. 2004; 15: 5712-5723
        • Borroni B.
        • Ghezzi S.
        • Agosti C.
        • Archetti S.
        • Fenoglio C.
        • Galimberti D.
        • Scarpini E.
        • Di Luca M.
        • Bresolin N.
        • Comi G.P.
        • Padovani A.
        • Del Bo R.
        Preliminary evidence that VEGF genetic variability confers susceptibility to frontotemporal lobar degeneration.
        Rejuvenation Res. 2008; 11: 773-780
        • Sleegers K.
        • Brouwers N.
        • Maurer-Stroh S.
        • van Es M.A.
        • Van Damme P.
        • van Vught P.W.
        • van der Zee J.
        • Serneels S.
        • De Pooter T.
        • Van den Broeck M.
        • Cruts M.
        • Schymkowitz J.
        • De Jonghe P.
        • Rousseau F.
        • van den Berg L.H.
        • Robberecht W.
        • Van Broeckhoven C.
        Progranulin genetic variability contributes to amyotrophic lateral sclerosis.
        Neurology. 2008; 71: 253-259
        • Jiao J.
        • Herl L.D.
        • Farese R.V.
        • Gao F.B.
        MicroRNA-29b regulates the expression level of human progranulin, a secreted glycoprotein implicated in frontotemporal dementia.
        PLoS One. 2010; 5: e10551
        • Rademakers R.
        • Eriksen J.L.
        • Baker M.
        • Robinson T.
        • Ahmed Z.
        • Lincoln S.J.
        • Finch N.
        • Rutherford N.J.
        • Crook R.J.
        • Josephs K.A.
        • Boeve B.F.
        • Knopman D.S.
        • Petersen R.C.
        • Parisi J.E.
        • Caselli R.J.
        • Wszolek Z.K.
        • Uitti R.J.
        • Feldman H.
        • Hutton M.L.
        • Mackenzie I.R.
        • Graff-Radford N.R.
        • Dickson D.W.
        Common variation in the miR-659 binding-site of GRN is a major risk factor for TDP43-positive frontotemporal dementia.
        Hum. Mol. Genet. 2008; 17: 3631-3642
        • Strong M.J.
        • Volkening K.
        • Hammond R.
        • Yang W.
        • Strong W.
        • Leystra-Lantz C.
        • Shoesmith C.
        TDP43 is a human low molecular weight neurofilament (hNFL) mRNA-binding protein.
        Mol. Cell. Neurosci. 2007; 35: 320-327
        • Irwin D.
        • Lippa C.F.
        • Rosso A.
        Progranulin (PGRN) expression in ALS. An immunohistochemical study.
        J. Neurol. Sci. 2009; 276: 9-13
        • Devos D.
        • Moreau C.
        • Lassalle P.
        • Perez T.
        • De Seze J.
        • Brunaud-Danel V.
        • Destée A.
        • Tonnel A.B.
        • Just N.
        Low levels of the vascular endothelial growth factor in CSF from early ALS patients.
        Neurology. 2004; 62: 2127-2129
        • Corrado L.
        • Ratti A.
        • Gellera C.
        • Buratti E.
        • Castellotti B.
        • Carlomagno Y.
        • Ticozzi N.
        • Mazzini L.
        • Testa L.
        • Taroni F.
        • Baralle F.E.
        • Silani V.
        • D'Alfonso S.
        High frequency of TARDBP gene mutations in Italian patients with amyotrophic lateral sclerosis.
        Hum. Mutat. 2009; 30: 688-694
        • Sun Z.
        • Diaz Z.
        • Fang X.
        • Hart M.P.
        • Chesi A.
        • Shorter J.
        • Gitler A.D.
        Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS.
        PLoS Biol. 2011; 9: e1000614
        • Wang J.W.
        • Brent J.R.
        • Tomlinson A.
        • Shneider N.A.
        • McCabe B.D.
        The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span.
        J. Clin. Invest. 2011; 121: 4118-4126
        • Lanson Jr., N.A.
        • Maltare A.
        • King H.
        • Smith R.
        • Kim J.H.
        • Taylor J.P.
        • Lloyd T.E.
        • Pandey U.B.
        A Drosophila model of FUS-related neurodegeneration reveals genetic interaction between FUS and TDP-43.
        Hum. Mol. Genet. 2011; 20: 2510-2523
        • Kryndushkin D.
        • Wickner R.B.
        • Shewmaker F.
        FUS/TLS forms cytoplasmic aggregates, inhibits cell growth, and interacts with TDP-43 in a yeast model of amyotrophic lateral sclerosis.
        Protein Cell. 2011; 2: 223-236
        • Anderson P.
        • Kedersha N.
        Stress granules. The Tao of RNA triage.
        Trends Biochem. Sci. 2008; 33: 141-150
        • Dormann D.
        • Rodde R.
        • Edbauer D.
        • Bentmann E.
        • Fischer I.
        • Hruscha A.
        • Than M.E.
        • Mackenzie I.R.
        • Capell A.
        • Schmid B.
        • Neumann M.
        • Haass C.
        ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import.
        EMBO J. 2010; 29: 2841-2857
        • Kim S.H.
        • Shanware N.P.
        • Bowler M.J.
        • Tibbetts R.S.
        Amyotrophic lateral sclerosis-associated proteins TDP-43 and FUS/TLS function in a common biochemical complex to co-regulate HDAC6 mRNA.
        J. Biol. Chem. 2010; 285: 34097-34105
        • Ling S.C.
        • Albuquerque C.P.
        • Han J.S.
        • Lagier-Tourenne C.
        • Tokunaga S.
        • Zhou H.
        • Cleveland D.W.
        ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 13318-13323
        • Wang X.
        • Arai S.
        • Song X.
        • Reichart D.
        • Du K.
        • Pascual G.
        • Tempst P.
        • Rosenfeld M.G.
        • Glass C.K.
        • Kurokawa R.
        Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription.
        Nature. 2008; 454: 126-130
        • Kovar H.
        Dr. Jekyll and Mr. Hyde. The two faces of the FUS/EWS/TAF15 protein family.
        Sarcoma. 2011; 2011: 837474
        • Fiesel F.C.
        • Voigt A.
        • Weber S.S.
        • Van den Haute C.
        • Waldenmaier A.
        • Görner K.
        • Walter M.
        • Anderson M.L.
        • Kern J.V.
        • Rasse T.M.
        • Schmidt T.
        • Springer W.
        • Kirchner R.
        • Bonin M.
        • Neumann M.
        • Baekelandt V.
        • Alunni-Fabbroni M.
        • Schulz J.B.
        • Kahle P.J.
        Knockdown of transactive response DNA-binding protein (TDP-43) down-regulates histone deacetylase 6.
        EMBO J. 2010; 29: 209-221
        • Van Damme P.
        • Van Hoecke A.
        • Lambrechts D.
        • Vanacker P.
        • Bogaert E.
        • van Swieten J.
        • Carmeliet P.
        • Van Den Bosch L.
        • Robberecht W.
        Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival.
        J. Cell Biol. 2008; 181: 37-41
        • Sathasivam S.
        VEGF and ALS.
        Neurosci. Res. 2008; 62: 71-77
        • Dormann D.
        • Haass C.
        TDP-43 and FUS: a nuclear affair.
        Trends Neurosci. 2011; (in press)
        • Gass J.
        • Prudencio M.
        • Stetler C.
        • Petrucelli L.
        Progranulin: An emerging target for FTLD therapies.
        Brain Res. 2012; (in press)
        • Levy N.S.
        • Chung S.
        • Furneaux H.
        • Levy A.P.
        Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR.
        J. Biol. Chem. 1998; 273: 6417-6423
        • Grillo G.
        • Turi A.
        • Licciulli F.
        • Mignone F.
        • Liuni S.
        • Banfi S.
        • Gennarino V.A.
        • Horner D.S.
        • Pavesi G.
        • Picardi E.
        • Pesole G.
        UTRdb and UTRsite (RELEASE 2010): a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs.
        Nucleic Acids Res. 2010; 38 (Database issue): D75-D80