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

Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 20149, Italy.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2012; 287(19):15635-47. DOI: 10.1074/jbc.M111.333450
Source: PubMed


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.

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Available from: Emanuele Buratti, Oct 10, 2015
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    • "Inthispicture(Figure6)FUSandTDP-43RNAbindingpro- teinsareatthecoreofthenetwork,sincetheyoftenareassociated factorsthatsharesimilarfeatures,withsometimesdifferentbut complementaryroles(Colombritaetal.,2012).Theyinteractwith RNAinthreemainroles:asRNAbindingproteinsparticipating onthedifferentaspectsofmRNAprocessing(Boscoetal.,2010; Colombritaetal.,2011),asregulatorsofmicroRNAsprocessing, "
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    ABSTRACT: Frontotemporal dementia (FTD) is a neurodegenerative disorder characterized by degeneration of the fronto temporal lobes and abnormal protein inclusions. It exhibits a broad clinicopathological spectrum and has been linked to mutations in seven different genes. We will provide a picture, which connects the products of these genes, albeit diverse in nature and function, in a network. Despite the paucity of information available for some of these genes, we believe that RNA processing and post-transcriptional regulation of gene expression might constitute a common theme in the network. Recent studies have unraveled the role of mutations affecting the functions of RNA binding proteins and regulation of microRNAs. This review will combine all the recent findings on genes involved in the pathogenesis of FTD, highlighting the importance of a common network of interactions in order to study and decipher the heterogeneous clinical manifestations associated with FTD. This approach could be helpful for the research of potential therapeutic strategies.
    Frontiers in Molecular Neuroscience 03/2015; 8:9. DOI:10.3389/fnmol.2015.00009 · 4.08 Impact Factor
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    • "For TDP43, GraphProt identifies a preference for repeated UG dinucleotides. TDP43 targets, determined by RNA immunoprecipitation followed by microarray analysis (RIP-chip), contained such repeats in 80% of the 3′ UTRs [43]. GraphProt motifs for PUM2, QKI and IGF2BP1-3 closely resemble the motifs previously identified using the same PAR-CLIP sets [6]. "
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    ABSTRACT: We present GraphProt, a computational framework for learning sequence- and structure-binding preferences of RNA-binding proteins (RBPs) from high-throughput experimental data. We benchmark GraphProt, demonstrating that the modeled binding preferences conform to the literature, and showcase the biological relevance and two applications of GraphProt models. First, estimated binding affinities correlate with experimental measurements. Second, predicted Ago2 targets display higher levels of expression upon Ago2 knockdown, whereas control targets do not. Computational binding models, such as those provided by GraphProt, are essential to predict RBP-binding sites and affinities in all tissues. GraphProt is freely available at
    Genome biology 01/2014; 15(1):R17. DOI:10.1186/gb-2014-15-1-r17 · 10.81 Impact Factor
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    • "An intriguing, but not mutually exclusive, possibility is that mutant-FUS and TDP-43 recruit additional protein partners and mRNA substrates into stress granules, thereby further increasing their size and abundance (Figure 4 and [25,30]). Indeed, thousands of mRNA transcripts are bound by FUS [58,59] with many distinct mRNAs bound by cytoplasmic mutant-FUS but not WT FUS [59]. Therefore, mutant-FUS may inappropriately process mRNAs and/or facilitate aberrant cytoplasmic protein interactions during stress. "
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    ABSTRACT: Amyotrophic lateral sclerosis (ALS)-linked fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS) is concentrated within cytoplasmic stress granules under conditions of induced stress. Since only the mutants, but not the endogenous wild-type FUS, are associated with stress granules under most of the stress conditions reported to date, the relationship between FUS and stress granules represents a mutant-specific phenotype and thus may be of significance in mutant-induced pathogenesis. While the association of mutant-FUS with stress granules is well established, the effect of the mutant protein on stress granules has not been examined. Here we investigated the effect of mutant-FUS on stress granule formation and dynamics under conditions of oxidative stress. We found that expression of mutant-FUS delays the assembly of stress granules. However, once stress granules containing mutant-FUS are formed, they are more dynamic, larger and more abundant compared to stress granules lacking FUS. Once stress is removed, stress granules disassemble more rapidly in cells expressing mutant-FUS. These effects directly correlate with the degree of mutant-FUS cytoplasmic localization, which is induced by mutations in the nuclear localization signal of the protein. We also determine that the RGG domains within FUS play a key role in its association to stress granules. While there has been speculation that arginine methylation within these RGG domains modulates the incorporation of FUS into stress granules, our results demonstrate that this post-translational modification is not involved. Our results indicate that mutant-FUS alters the dynamic properties of stress granules, which is consistent with a gain-of-toxic mechanism for mutant-FUS in stress granule assembly and cellular stress response.
    Molecular Neurodegeneration 08/2013; 8(1):30. DOI:10.1186/1750-1326-8-30 · 6.56 Impact Factor
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