Buratti, E. et al. 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. J. Biol. Chem. 280, 37572-37584. A study of the interaction between the C terminus of TDP43 and other members of the hnRNP complex, including hnRNP A2/B1 and hnRNP A1. This paper also shows that TDP43 regulates splicing in the early stages of spliceosomal assembly

International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2005; 280(45):37572-84. DOI: 10.1074/jbc.M505557200
Source: PubMed


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.

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    • "Moreover, the pathological mutations conducted in this study were located in the glycine-rich domain (residues 274–314), whereas this region has been reported structurally similar to heterogeneous nuclear ribonuclear proteins (hnRNPs) [36], [37]. The interaction between TDP-43 and hnRNPA2 is important in maintaining cellular homeostasis while alterations in TDP-43-recruited hnRNP complex formation may perturb downstream RNA splicing regulation and protein-protein interactions [38], [39]. Future experiments will be applied to dissect how structural aberrations and prion-like properties in TDP C-terminus mutants (G294V and G295S) influence the interaction with their downstream cellular binding partners in TDP-43 proteinopathy. "
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    ABSTRACT: TAR DNA-binding protein (TDP-43) was identified as the major ubiquitinated component deposited in the inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) in 2006. Later on, numerous ALS-related mutations were found in either the glycine or glutamine/asparagine-rich region on the TDP-43 C-terminus, which hinted on the importance of mutations on the disease pathogenesis. However, how the structural conversion was influenced by the mutations and the biological significance of these peptides remains unclear. In this work, various peptides bearing pathogenic or de novo designed mutations were synthesized and displayed their ability to form twisted amyloid fibers, cause liposome leakage, and mediate cellular toxicity as confirmed by transmission electron microscopy (TEM), circular dichroism (CD), Thioflavin T (ThT) assay, Raman spectroscopy, calcein leakage assay, and cell viability assay. We have also shown that replacing glycines with prolines, known to obstruct β-sheet formation, at the different positions in these peptides may influence the amyloidogenesis process and neurotoxicity. In these cases, GGG308PPP mutant was not able to form beta-amyloid, cause liposome leakage, nor jeopardized cell survival, which hinted on the importance of the glycines (308-310) during amyloidogenesis.
    PLoS ONE 08/2014; 9(8):e103644. DOI:10.1371/journal.pone.0103644 · 3.23 Impact Factor
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    • "As shown in Figure 1A, TDP-43 contains two RNA recognition motifs that are involved in its function in RNA stabilization and processing, while the carboxy-terminus is believed to drive a toxic gain of function, as the majority of ALS and FTLD-TDP-linked mutations are found in this glycine-rich region of the protein [3], [4], [5], [6]. This carboxy-terminal portion of the TDP-43 molecule shares homology with members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and can bind hnRNP A/B and hnRNP A1 [7]. In addition, the C-terminus appears to contain prion-like sequences that can promote aggregation and stress granule formation, potentially reducing its normal RNA functional activities [8], [9], [10], [11]. "
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    ABSTRACT: Trans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25-50% of Alzheimer's Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10-12 nm filaments though wider 18-20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades. The protocols described here generate soluble, full-length and untagged TDP-43 allowing for a direct assessment of the impact of various posttranslational modifications on TDP-43 function. We demonstrate that Casein Kinase II (CKII) promotes the polymerization of this soluble TDP-43 into 10 nm diameter filaments that resemble the most common TDP-43 structures observed in disease. Furthermore, these filaments are recognized as abnormal by Heat Shock Proteins (HSPs) which can inhibit TDP-43 polymerization or directly promote TDP-43 filament depolymerization. These findings demonstrate CKII induces polymerization of soluble TDP-43 into filaments and Hsp90 promotes TDP-43 filament depolymerization. These findings provide rational for potential therapeutic intervention at these points in TDP-43 proteinopathies.
    PLoS ONE 03/2014; 9(3):e90452. DOI:10.1371/journal.pone.0090452 · 3.23 Impact Factor
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    • "TAR DNA binding protein of 43 kilodaltons (TDP-43) is the major pathological substrate in frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and most cases of amyotrophic lateral sclerosis (ALS) [1]. TDP-43 is a member of the hnRNP family, containing two RNA Recognition Motifs (RRMs) that bind RNA and a C-terminal, glycine rich domain that mediates interactions with functional binding partners to coordinate the metabolism of a wide range of RNA substrates [2], [3]. In disease, normal nuclear localization of TDP-43 is lost, and ubiquitinated and hyperphosphorylated inclusions are deposited in the form of neuronal intranuclear inclusions or in the cytoplasm as juxtanuclear aggregates or dystrophic neurites [4]. "
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    ABSTRACT: The majority of cases of frontotemporal lobar degeneration and amyotrophic lateral sclerosis are pathologically defined by the cleavage, cytoplasmic redistribution and aggregation of TAR DNA binding protein of 43 kDa (TDP-43). To examine the contribution of these potentially toxic mechanisms in vivo, we generated transgenic mice expressing human TDP-43 containing the familial amyotrophic lateral sclerosis-linked M337V mutation and identified two lines that developed neurological phenotypes of differing severity and progression. The first developed a rapid cortical neurodegenerative phenotype in the early postnatal period, characterized by fragmentation of TDP-43 and loss of endogenous murine Tdp-43, but entirely lacking aggregates of ubiquitin or TDP-43. A second, low expressing line was aged to 25 months without a severe neurodegenerative phenotype, despite a 30% loss of mouse Tdp-43 and accumulation of lower molecular weight TDP-43 species. Furthermore, TDP-43 fragments generated during neurodegeneration were not C-terminal, but rather were derived from a central portion of human TDP-43. Thus we find that aggregation is not required for cell loss, loss of murine Tdp-43 is not necessarily sufficient in order to develop a severe neurodegenerative phenotype and lower molecular weight TDP-43 positive species in mouse models should not be inherently assumed to be representative of human disease. Our findings are significant for the interpretation of other transgenic studies of TDP-43 proteinopathy.
    PLoS ONE 01/2014; 9(1):e86513. DOI:10.1371/journal.pone.0086513 · 3.23 Impact Factor
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