Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery

Abstract

TDP-43 is a highly conserved and ubiquitously expressed member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins. Recently, TDP-43 was shown to be a major disease protein in the ubiquitinated inclusions characteristic of most cases of amyotrophic lateral sclerosis (ALS), tau-negative frontotemporal lobar degeneration (FTLD), and inclusion body myopathy. In these diseases, TDP-43 is redistributed from its predominantly nuclear location to ubiquitin-positive, cytoplasmic foci. The extent to which TDP-43 drives pathophysiology is unknown, but the identification of mutations in TDP-43 in familial forms of ALS and FTLD-U suggests an important role for this protein in pathogenesis. Little is known about TDP-43 function and only a few TDP-43 interacting proteins have been previously identified, which makes further insight into both the normal and pathological functions of TDP-43 difficult. Here we show, via a global proteomic approach, that TDP-43 has extensive interaction with proteins that regulate RNA metabolism. Some interactions with TDP-43 were found to be dependent on RNA-binding, whereas other interactions are RNA-independent. Disease-causing mutations in TDP-43 (A315T and M337V) do not alter its interaction profile. TDP-43 interacting proteins largely cluster into two distinct interaction networks, a nuclear/splicing cluster and a cytoplasmic/translation cluster, strongly suggesting that TDP-43 has multiple roles in RNA metabolism and functions in both the nucleus and the cytoplasm. Finally, we found numerous TDP-43 interactors that are known components of stress granules, and indeed, we find that TDP-43 is also recruited to stress granules.

Figure 1. Identification of TDP-43 interacting proteins by FLAG-immunoprecipitation

(A) Immunoprecipitates from FLAG-TDP-43-expressing HEK-293T cells or control HEK-293T cells were separated by gel electrophoresis and stained with Sypro-Ruby to visualize proteins. Both the control and FLAG-TDP-43 lanes were separated into 24 bands along the entire length of the gel and analyzed by mass spectrometry. Intervening empty lanes were removed for visualization purposes. (B) Pie-chart representation of functional classes of TDP-43-interacting proteins.

Figure 2. TDP-43 interacting proteins form two distinct protein interaction networks

TDP-43 proteins identified by mass spectrometry were analyzed using STRING interaction software to identify high confidence interactions using database, literature, and experimental search parameters. Only proteins that were at least two-fold enriched in the TDP-43 immunoprecipitate were analyzed using STRING. Two distinct protein interactions were observed that are labeled as the Nuclear/Splicing Cluster and the Cytoplasmic/Translation Cluster.

Figure 3. The impact of TDP-43 mutations on interactions

(A) Disease-associated mutations do not alter the TDP-43 interactome. The figure shows Sypro-Ruby-stained FLAG immunoprecipitates from control HEK-293T cells, HEK-293T cells expressing wild type FLAG-TDP-43, FLAG-TDP-43 (A315T) or FLAG-TDP-43 (M337V) as indicated. FLAG-TDP-43 (M337V) reproducibly immunoprecipitates less efficiently than either FLAG-TDP-43 or FLAG-TDP-43 (A315T) which is proportional to the decrease in intensity of interacting proteins as visualized by Sypro-Ruby. (B) Some TDP-43 interactions are RNA-dependent. The figure shows Sypro-Ruby-stained FLAG immunoprecipitates from control HEK-293T cells, HEK-293T cells expressing wild type FLAG-TDP-43, wild type FLAG-TDP-43 (treated with RNase A), or FLAG-TDP-43 (mutRRM), as indicated. Immunoprecipitation was repeated at least three times with consistent results. Representative images were chosen for display.

Figure 4. Characterization of TDP-43 interaction with hnRNP H and PABPC1

(A) Validation of TDP-43 interaction with hnRNP H and PABPC1 by co-immunoprecipitation followed by Western blot analysis in HEK-293T cells. Left panel: Western blot analysis of whole cell lysates prior to immunoprecipitation was used to visualize 1% of protein input. Right panel: Western blot analysis of FLAG immunoprecipitates. Quantification was performed using Image J (shown below each band) and normalized to the amount of TDP-43 in lane 2. Immunoprecipitation was repeated at least three times with consistent results and representative images were chosen for display. (B) Immunofluorescence was used to visualize the localization of TDP-43 and hnRNP H in HeLa cells. DAPI staining was used to visualize the nucleus. TDP-43 and hnRNP H both showed pan-nuclear expression with co-localization in sub-nuclear foci in HeLa cells. The immunofluorescence data shown represents consistent results obtained in multiple replicates. IB: immunoblot, IP: immunoprecipitation.

Figure 5. Cytoplasmic TDP-43 is localized in stress granules

Immunofluorescence was used to visualize the localization of exogenous (A-C) FLAG-TDP-43 or endogenous (D) TDP-43 and (A) PABPC1, (B, D) G3BP1 and (C) EIF4G in HeLa cells. DAPI staining was used to visualize the nucleus. TDP-43 was found to localize with stress granules in the cytoplasm of HeLa cells. (D) After treatment with 50 μM MG-132 for 3 hours, RNA granules were observed in 66% of cells. At least 1 TDP-43 positive stress granule was observed in 25% of cells after MG-132 treatment. 300 HeLa cells were counted. All of the immunofluorescence data shown represents consistent results obtained in multiple replicates.

Figure 6. TDP-43 association with stress granules is strongly mitigated by inability to bind RNA

(A) TDP-43(mutRRM) was rarely found in cytoplasmic RNA granules (as visualized by EIF4G). (B) In FLAG-TDP-43 expressing cells, FLAG-TDP-43 was found to co-localize with EIF4G in 85% of stress granules (n=242 cells) whereas FLAG-TDP-43(mutRRM) was found in only 6.5% of stress granules (n=168 cells).