TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail - An important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing

International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Journal of Biological Chemistry (Impact Factor: 4.6). 12/2005; 280(45):37572-84. DOI: 10.1074/jbc.M505557200
Source: PubMed

ABSTRACT TDP-43 is a highly conserved nuclear factor of yet unknown function that binds to ug-repeated sequences and is responsible for cystic fibrosis transmembrane conductance regulator exon 9 splicing inhibition. We have analyzed TDP-43 interactions with other splicing factors and identified the critical regions for the protein/protein recognition events that determine this biological function. We show here that the C-terminal region of TDP-43 is capable of binding directly to several proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with well known splicing inhibitory activity, in particular, hnRNP A2/B1 and hnRNP A1. Mutational analysis showed that TDP-43 proteins lacking the C-terminal region could not inhibit splicing probably because they were unable to form the hnRNP-rich complex involved in splicing inhibition. Finally, through splicing complex analysis, we show that splicing inhibition mediated by TDP-43 occurs at the earliest stages of spliceosomal assembly.


Available from: Emanuele Buratti, Jun 12, 2015
  • Source
  • [Show abstract] [Hide abstract]
    ABSTRACT: Fragile X-associated Tremor Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by a CGG trinucleotide repeat expansion in the 5'UTR of the Fragile X gene, FMR1. FXTAS is thought to arise primarily from an RNA gain-of-function toxicity mechanism. However, recent studies demonstrate that the repeat also elicits production of a toxic polyglycine protein, FMRpolyG, via Repeat-Associated Non-AUG (RAN) initiated translation. Pathologically, FXTAS is characterized by ubiquitin positive intranuclear neuronal inclusions, raising the possibility that failure of protein quality control pathways could contribute to disease pathogenesis. To test this hypothesis, we used Drosophila and cell based models of CGG-repeat associated toxicity. In Drosophila, ubiquitin proteasome system (UPS) impairment led to enhancement of CGG-repeat induced degeneration whereas overexpression of the chaperone protein HSP70 suppressed this toxicity. In transfected mammalian cells, CGG-repeat expression triggered accumulation of a GFP-UPS reporter in a length-dependent fashion. To delineate the contributions from CGG repeats as RNA from RAN translation associated toxicity, we enhanced or impaired the production of FMRpolyG in these models. Driving expression of FMRpolyG enhanced induction of UPS impairment in cell models while prevention of RAN translation attenuated UPS impairment in cells and suppressed the genetic interaction with UPS manipulation in Drosophila. Taken together, these findings suggest that CGG repeats induce ubiquitin proteasome system impairment at least in part through activation of RAN translation. Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
    Human Molecular Genetics 05/2015; DOI:10.1093/hmg/ddv165 · 6.68 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We have previously demonstrated that infection by coxsackievirus B3 (CVB3), a positive-stranded RNA enterovirus, results in the accumulation of insoluble ubiquitin–protein aggregates, which resembles the common feature of neurodegenerative diseases. The importance of protein aggregation in viral pathogenesis has been recognized; however, the underlying regulatory mechanisms remain ill-defined. Transactive response DNA-binding protein-43 (TDP-43) is an RNA-binding protein that has an essential role in regulating RNA metabolism at multiple levels. Cleavage and cytoplasmic aggregation of TDP-43 serves as a major molecular marker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration and contributes significantly to disease progression. In this study, we reported that TDP-43 is translocated from the nucleus to the cytoplasm during CVB3 infection through the activity of viral protease 2A, followed by the cleavage mediated by viral protease 3C. Cytoplasmic translocation of TDP-43 is accompanied by reduced solubility and increased formation of protein aggregates. The cleavage takes place at amino-acid 327 between glutamine and alanine, resulting in the generation of an N-and C-terminal cleavage fragment of ~ 35 and ~ 8 kDa, respectively. The C-terminal product of TDP-43 is unstable and quickly degraded through the proteasome degradation pathway, whereas the N-terminal truncation of TDP-43 acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Lastly, we demonstrated that knockdown of TDP-43 results in an increase in viral titers, suggesting a protective role for TDP-43 in CVB3 infection. Taken together, our findings suggest a novel model by which cytoplasmic redistribution and cleavage of TDP-43 as a consequence of CVB3 infection disrupts the solubility and transcriptional activity of TDP-43. Our results also reveal a mechanism evolved by enteroviruses to support efficient viral infection. Cell Death and Differentiation advance online publication, 15 May 2015; Coxsackievirus B3 (CVB3) is a small, positive-stranded RNA enterovirus. 1 The single open reading frame of CVB3 is translated into a viral polypeptide that is subsequently cleaved by two virus-encoded proteases 2A and 3C to generate structural and non-structural proteins. 2 In addition to processing viral polyprotein, 2A and 3C target host proteins important for maintenance of protein translation and transcription, antiviral activity, and cellular architecture and signaling, contributing to virus-induced pathogenesis. 3–5 Although enteroviral replication takes place exclusively in the cyto-plasm, viral infection has been demonstrated to lead to cytoplasmic translocation of nuclear proteins. 6 For example, heterogeneous ribonucleoprotein D (hnRNP D) has been shown to translocate from the nucleus to the cytoplasm during enteroviral infection. 5,7,8 Moreover, hnRNP D is cleaved by 3C and has an antiviral function against enteroviral infection. 5,7,8
    Cell Death and Differentiation 05/2015; DOI:10.1038/cdd.2015.58 · 8.39 Impact Factor