The N-terminal dimerization is required for TDP-43 splicing activity - PubMed (original) (raw)

The N-terminal dimerization is required for TDP-43 splicing activity

Lei-Lei Jiang et al. Sci Rep. 2017.

Abstract

TDP-43 is a nuclear factor that functions in promoting pre-mRNA splicing. Deletion of the N-terminal domain (NTD) and nuclear localization signal (NLS) (i.e., TDP-35) results in mislocalization to cytoplasm and formation of inclusions. However, how the NTD functions in TDP-43 activity and proteinopathy remains largely unknown. Here, we studied the structure and function of the NTD in inclusion formation and pre-mRNA splicing of TDP-43 by using biochemical and biophysical approaches. We found that TDP-43 NTD forms a homodimer in solution in a concentration-dependent manner, and formation of intermolecular disulfide results in further tetramerization. Based on the NMR structure of TDP-43 NTD, the dimerization interface centered on Leu71 and Val72 around the β7-strand was defined by mutagenesis and size-exclusion chromatography. Cell experiments revealed that the N-terminal dimerization plays roles in protecting TDP-43 against formation of cytoplasmic inclusions and enhancing pre-mRNA splicing activity of TDP-43 in nucleus. This study may provide mechanistic insights into the physiological function of TDP-43 and its related proteinopathies.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Figure 1

Characterization of the oligomeric states of the N-terminal fragments of TDP-43 by SEC. (A) Schematic representation of the domain architecture of TDP-43 and its N-terminal fragments. NTD, N-terminal domain; NLS, nuclear localization signal; RRM, RNA recognition motif; GRR, glycine-rich region; AC, amyloidogenic core. (B) SEC analysis of the N-terminal fragments of TDP-43. The proteins were diluted with Buffer A (25 mM Tris-HCl, pH 8.0, and 150 mM NaCl) to a concentration of 50 µM and loaded onto a Superdex-200 Increase 10/30 GL column. The chromatographic profiles are shown for TDP(1–261) (black), TDP(1–89) (red), and TDP(101–261) (blue). A280nm, Absorbance at 280 nm; mAU, micro absorbance unit. (C) Standard profile for calculating the apparent molecular weight (MW). Ve/V0, relative elution volume. The standard markers are: BSA (67.0 kDa), GST (52.6 kDa), ovobumin (44.3 kDa), thioredoxin (13.9 kDa), and cytochrome C (12.4 kDa). The concentrations of the standards are around 10 to 100 μM. (D) As in (B), SEC profiles of the N-terminal fragments of TDP-43 at 10-µM concentration.

Figure 2

Figure 2

SEC profiles for TDP(1–77) and its Cys mutant. (A) SEC profiles for TDP(1–77) at different concentrations. (B) SEC profiles for TDP(1–77) with (pink) or without 5 mM DTT (black). The protein concentration was to 500 µM. (C) SEC profiles for TDP(1–77)-C39/C50S at different concentrations. The SEC experiments were carried out using a Superdex-200 Increase 10/30 GL column previously equilibrated with Buffer A. The right panels show the normalized chromatograms.

Figure 3

Figure 3

Solution structure of TDP(1–77) (C39/C50S) elucidated by NMR. (A) Superposition of the backbone traces of the 10 lowest-energy structures. (B) Ribbon diagram of a representative structure of TDP(1–77) (C39/C50S), showing seven β-strands and one short α-helix. The structure was elucidated with the GB1-fused Cys mutant TDP(1–77)-GB1-C39/C50S. (C) Comparison of the overall structures of TDP(1–77) from TDP(1–77)-GB1-C39/C50S (blue) and His6-TDP(1–77) (pink; PDB: 2N4P). The RMSD is 2.96 Å. N, N-terminus; C, C-terminus. All the structures are displayed using MOLMOL.

Figure 4

Figure 4

Identification of the dimer interfaces of TDP(1–77) by mutagenesis and SEC. (A) SEC profiles for TDP(1–77)-GB1-C39/C50S. (B) L27/I60/V57A mutant. (C) L71/Y73A mutant. (D) V72/V74/Y43A mutant. The right panels show the normalized chromatograms. The mutants were generated based on TDP(1–77)-GB1-C39/C50S. The proteins were diluted to the indicated concentrations with Buffer A and loaded onto a Superdex-75 10/30 GL column.

Figure 5

Figure 5

Immunofluorescence microscopic imaging of cytoplasmic inclusions of TDP-43-NLSmut and its dimer-interface mutants. (A) Inclusion formation of FLAG-TDP-43-NLSmut and its Cys mutant (C39/C50S) and dimer-interface mutants (CS-L71/Y73A and CS-V72/V74/Y43A) in HEK 293 T cells. TDP-43-NLSmut and its mutants were stained with mouse anti-FLAG (green), and the nuclei were stained with Hoechst (blue). The images are shown with a magnification of 3 times (3 X). Scale bar = 10 µm. The mutants were generated based on FLAG-TDP-43-NLSmut. (B) Quantification of the cells with cytoplasmic inclusions in HEK 293 T cells overexpressing FLAG-TDP-43-NLSmut or its mutants. Cells were counted and the data were statistically analyzed by one-way ANOVA. Results are presented as Mean ± SEM (n = 25–30). ***p < 0.001; n.s., no significance.

Figure 6

Figure 6

Effects of dimer-interface mutations on the RNA splicing activity of TDP-43. (A) Effects of TDP-43, its Cys mutant (C39/C50S) and dimer-interface mutants (CS-L71/Y73A and CS-V72/V74/Y43A) on CFTR exon 9 splicing. The GT13T5 reporter was co-transfected with the FLAG-tagged plasmids into HEK 293 T cells, respectively. Exon 9 + stands for unspliced cDNA fragment, while exon 9− denotes spliced cDNA. (B) Quantification of the spliced RT-PCR products of TDP-43 and its mutants based on band intensity. Data were analyzed by one-way ANOVA and represented as Mean ± SD (n = 3). ***p < 0.001. (C) Immunofluorescence microscopic images showing nuclear localization of FLAG-TDP-43 and its mutants in HEK 293 T cells. TDP-43 and its mutants were stained with mouse anti-TDP-43 or anti-FLAG (green), and the nuclei were stained with Hoechst (blue). The images are shown with a magnification of 3 times (3 X). Scale bar = 10 µm. (D) Western blot showing the expression levels of the protein species for CFTR splicing assay.

Figure 7

Figure 7

Schematic representation for the oligomerization of TDP-43. The model proposes that TDP-43 NTD mainly forms a dimer via residues Leu71 and Val72 around the β7-strand, and then transforms into a tetramer via disulfide formation at high protein concentrations. The NTD dimerization or tetramerization may protect TDP-43 against aggregation and is required for splicing activity of TDP-43.

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