The N-terminal dimerization is required for TDP-43 splicing activity
SCIEntIfIC REPORTs |
The N-terminal dimerization is required for TDP-43 splicing activity
OPEN 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 TDP43 NTD, the dimerization interface centered on Leu71 and Val72 around the ?7-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.
TDP-43 (TAR DNA-binding protein of 43 kDa) is a DNA/RNA binding protein belonging to the heterogeneous
nuclear ribonucleoprotein (hnRNP) family. It was originally identified as a transcriptional repressor of the HIV-1
gene1. TDP-43 is highly conserved from human to Caenorhabditis elegans2 and plays multiple roles in
transcription, pre-mRNA splicing and processing, regulation of translation, and RNA stability3?6. From its primary
structure, TDP-43 is composed of an N-terminal domain (NTD), two RNA recognition motifs (RRMs), and a long
C-terminal glycine-rich region (GRR) (see Fig.?1A). The RRM domains are responsible for binding to UG and TG
repeats of RNA and DNA2, 7, whereas the GRR mediates protein-protein interactions between TDP-43 and other
hnRNP members8, 9. TDP-43 also contains a nuclear localization signal (NLS) and a nuclear export signal (NES),
shuttling TDP-43 between the nucleus and the cytoplasm10, 11.
So far, TDP-43 has been shown to be the major component of inclusions associated with neurodegenerative
diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD)12, 13. The
disease-specific biochemical signatures of TDP-43 have been studied worldwide10, 12, 13. Under physiological
conditions, TDP-43 localizes primarily in nucleus. However, it undergoes ubiquitination, redistribution from the
nucleus to cytoplasm, N-terminal truncation and formation of cytoplasmic inclusions, and consequently loses
its normal function in pathological cases, known as TDP-43 proteinopathies10, 12?15. The pathological mechanism
induced by TDP-43 has been reviewed broadly16?19. In disease pathologies, the C-terminal fragments have been
investigated considerably; they are essential for TDP-43 aggregation and inclusion formation20?26. Some studies
have defined the Gln/Asn-rich domain27, 28 or amyloidogenic cores29?31 in the C-terminal region for TDP-43
aggregation. In addition, numerous mutations in TDP-43, mainly in the C-terminal region, have been identified
in familial and sporadic cases of ALS and FTLD, and demonstrated to influence the aggregation and cellular
function of TDP-4314, 32?36 and to alter axonal transportation37, 38.
Several studies have suggested that TDP-43 mainly forms a functional homodimer under physiological
conditions, but it may transform to oligomers or aggregates, which lead to loss of function in pathological diseases39?43.
To date, accumulating evidence has directed the research focusing on the importance of the C-terminal part for
TDP-43 aggregation, whereas the biological and pathological roles of the N-terminus have been studied to a
limited extent. In the past few years, some studies have revealed that the structure and structural integrity of the NTD
are essential for its self-interaction and biological function44?46. Very recently, the solution structures of TDP-43
NTD have been elucidated by NMR under extreme conditions47, 48 and thoroughly compared and evaluated49.
They are both canonical ?-barrel structures constructed by seven ?-strands and a short ?-helix. In this regard,
mechanistic study based on structural analysis of the NTD will be beneficial to understanding the biological roles
played by TDP-43 under physiological and pathological conditions. In this communication, we further
investigated the biochemical properties and physiological function of the NTD of TDP-43. We found that TDP-43 NTD
forms a dimer or tetramer under normal conditions and inhibits TDP-43 aggregation. Disruption of dimerization
can result in loss of TDP-43 splicing activity. This study may provide further insights into the biological function
of the N-terminal dimerization in integration of TDP-43 conformation and its splicing activity, which is
implicated in the pathological roles in neurodegenerative diseases.
The N-terminal domain of TDP-43 forms a homodimer. It was proposed that TDP-43 forms a dimeric
structure under physiological conditions and its NTD is capable of oligomerization and self-interaction42?48. Our
previous study revealed that staurosporine (STS)-triggered removal of the N-terminus (including NTD and NLS)
from TDP-43 (i.e., TDP-35) causes cytoplasmic localization and inclusion formation20. To obtain further insight
into the physiological properties of TDP-43 NTD, we constructed different N-terminal fragments of TDP-43
(Fig.?1A) and performed size-exclusion chromatography (SEC) experiments (Fig.?1). As a result, the retention
volume of the main peak of TDP(
) at a protein concentration of 50 ?M was 13.92 mL (Fig.?1B), corresponding
to an apparent molecular weight (MW) of 68.6 kDa as estimated based on the standard proteins (Fig.?1C). This
apparent MW was close to the theoretical value for a dimer (2 ? 30.7 kDa) (Table?1). The retention volume of the
main peak of TDP(
) at the same concentration was 16.27 mL, corresponding to an MW of 27.1 kDa, also
estimated to be a dimer. However, the apparent MW of TDP(101?261) was close to the theoretical value, suggesting
that it forms a monomer in solution (Table?1). Considering the normal concentration of TDP-43 in cell and the
limitation of detecting low-concentration proteins in SEC experiment, we determined the retention volume of
the TDP-43 fragments at a 10-?M concentration. The three TDP-43 fragments exhibited elution profiles similar
to the respective ones at the 50-?M concentration (Fig.?1D). We also observed the retention peaks of TDP(
at different concentrations (Fig.?S1A). With the increase of protein concentration, the retention peak gradually
shifted to that of a larger MW, suggesting that TDP(
) altered its state from dimer to tetramer at a higher
) had a similar SEC profile to TDP(
) (Fig.?S1B). However, TDP(101?261)
exhibited almost the same retention volumes at different concentrations (Fig.?S1C), suggesting that it forms a monomer
in solution. Thus, the results from SEC experiments demonstrate that TDP-43 NTD is the source of dimerization
and even further tetramerization of TDP-43.
Formation of intermolecular disulfide promotes further tetramerization of TDP-43 NTD. In
the SEC experiments, we also observed an additional minor peak corresponding to a larger MW oligomer eluted
out before the major peak in the chromatograms for TDP(
) (Fig.?S2A) and TDP(
) (Fig.?S2B). However,
when treated with 5 mM DTT, the minor peaks disappeared, suggesting that these large MW oligomers formed
through intermolecular disulfides. On the contrary, DTT treatment had no effect on the monomeric state of
TDP(101?261) (Fig.?S2C). Therefore, we presumed that the Cys residues in TDP-43 NTD contribute to the
oligomerization of TDP-43. To exclude the influence of disulfide formation on the oligomerization of TDP-43 NTD,
we mutated both Cys39 and Cys50 to Ser residues. We observed that the additional peaks disappeared, as in the
case of DTT treatment (Fig.?S2D,E). Similar to that of the wild type, the main peaks of TDP(
) and TDP(
) mutants also had small shifts with increasing concentrations, but they still remained as dimers. Collectively,
formation of the intermolecular disulfides by Cys residues is beneficial for tetramerization of TDP-43 NTD.
) is a stable dimer in solution. Because TDP(
) is not stable in solution, we prepared the
N-terminal 77-residue construct (TDP(
)) for further structural analysis. With the increase of protein
concentration, the SEC profile of TDP(
) had two peaks, a major and a minor peak (Fig.?2A). We estimated that
the major peaks reflect the dimeric states, whereas the minor peaks correspond to the tetrameric states (Table?2).
Moreover, the minor peak vanished when the sample was treated with 5 mM DTT (Fig.?2B). Interestingly, a Cys
mutant (C39/C50S) of TDP(
) exhibited only one sharp peak at any concentration tested (Fig.?2C). The
retention volume of the peak was ~17.6 mL corresponding to an MW of 16.0 kDa, suggesting that only the dimeric
state existed in the Cys mutant.
We next performed dynamic light scattering (DLS) analysis on TDP(
) and its Cys mutant to verify their
size and size distribution (Fig.?S3). TDP(
) gave a wide size distribution of roughly spherical particles with an
average hydrodynamic radius of 3.1 nm and an apparent MW of ~47 kDa, suggesting that wild-type TDP(
formed a large population of tetramers in solution. This is consistent with the previous observation from the SEC
experiments (Table?2) that the particle size was gradually enlarged with the increase of protein concentration.
However, the size distribution of TDP (
)-C39/C50S exhibited a single narrow peak in the DLS intensity
profile. The average hydrodynamic radius was 2.3nm and the apparent MW was ~21 kDa, strongly supporting
formation of a stable dimer of the Cys mutant.
We also carried out non-reducing SDS-PAGE analysis to examine the effect of the two Cys residues on the
oligomeric state of TDP-43 NTD. TDP(
) and its two single-Cys mutants (C39S, C50S) presented a band
corresponding to the dimeric state in the absence of DTT, whereas the double mutant (C39/C50S) exhibited no
band for dimeric state whether DTT was present or not (Fig.?S4A). Based on the band intensity, Cys50 may be
more accessible and prone to intermolecular disulfide formation than Cys39. This suggests that
intermolecular disulfide formation contributes to dimerization or tetramerization. Circular dichroism (CD) spectroscopy
indicated that disrupting the disulfide-induced oligomerization had little effect on the secondary structures of
) (Fig.?S4B), although all TDP(
) variants gave the non-canonical CD spectra. Taken together,
these biochemical results confirm that TDP-43 NTD is able to form dimers itself and further tetramers through
disulfide formation, which is sufficient for leading full-length TDP-43 to dimerization and tetramerization.
Solution structure of TDP-43 NTD. To gain further insights into the dimerization of TDP-43 NTD, we
solved its structure in solution by using NMR techniques. In order to improve its stability and homogeneity for
NMR data acquisition, we fused GB1 to the C-terminus of TDP(
)-GB1). Meanwhile, we also
applied SEC, DLS and non-reducing SDS-PAGE to confirm the stability and homogeneity of TDP(
and its Cys mutant (Fig.?S5). TDP(
)-GB1 exhibited similar biochemical characteristics to TDP(
supporting the existence of dimers with a small population of tetramers in solution (Fig.?S5A,B,D). As the Cys
residues (C39, C50) contribute to the formation of tetramer from dimer, which may influence the
homogeneity of the protein, we applied a Cys mutant (TDP(
)-GB1-C39/C50S) to structural analysis by NMR. As
)-GB1-C39/C50S was still in the stable dimeric state under an NMR condition (Fig.?S5C,E).
We assigned the chemical shifts and unambiguous intramolecular NOEs to calculate the monomeric structure.
The restraints used for structure calculation and statistics are summarized in Table?S1. In general, TDP(
forms a ?-barrel structure mainly comprised of two ?-sheets (?1?2?4?7, ?3?5?6) with a short ?-helix between
them (Fig.?3A,B). Overall, it shows structural fold and secondary-structure pattern similar to the previously
reported structure (PDB ID: 2N4P) solved in dilute protein concentration48, giving an RMSD of 2.96 ?. Besides
some difference in the flexible regions, the ?-helix and ?3-strand are two-residue shorter than those of 2N4P,
whereas the ?6-strand is two-residue longer. However, this structure is largely different from that solved under
the pure-water condition47.
The dimeric interface of TDP-43 NTD. TDP-43 NTD forms predominantly dimeric structures under
normal conditions, but the residues that are crucial for dimerization are unknown. To address this issue, we
prepared several mutants based on the monomeric structure (Fig.?3) and the predicted interfaces of TDP(
and performed SEC analysis (Fig.?4). As a result, TDP(
)-GB1-C39/C50S eluted with a single peak
corresponding to an apparent MW of 25.6 kDa (Fig.?4A), suggesting it formed a folded dimer. The L27A/I60A/V57A
mutant (mutation around the ?-helix) was well in line with TDP(
)-GB1-C39/C50S (Fig.?4B), also eluted as
an apparent dimer. However, additional peaks corresponding to monomers eluted after the main peaks both for
L71A/Y73A and V72A/V74A/Y43A mutants (mutation mainly in the ?7 strand) at low protein concentrations,
500 + DTT
implying that these two mutants had their dimers partly dissociated to monomers (Fig.?4C,D). These results
suggest that the residues of Leu71-Val74 in the ?7-strand are mainly clustered in the dimer interface, and they are
crucial for TDP-43 NTD dimerization.
Because CD spectroscopy could not provide secondary structure information for TDP (
) (Fig.?S4B), we
resorted to Fourier transform infrared (FT-IR) spectroscopy to compare the secondary structures of TDP (
and its mutants. Quantitative analysis revealed that the ?-sheet contents of the L71/Y73A and V72/V74/Y43A
mutants were roughly unchanged, as compared with those of TDP(
) and its C39/C50S mutant (Fig.?S6).
Moreover, the secondary structure content of TDP(
)-C39/C50S estimated from FT-IR was consistent with
that calculated from our NMR structure (PDB ID: 5X4F). These results suggest that transformation of the
oligomeric states, tetramer to dimer to monomer, has no considerable influence on the secondary structures of
The N-terminal dimerization inhibits formation of TDP-43 inclusions in cytoplasm. Our
previous study revealed that TDP-35 mislocalizes in the cytoplasm and forms inclusions due to removal of the NLS
and NTD. To further obtain information of TDP-43 NTD dimerization in cytoplasmic inclusion formation, we
prepared Cys-to-Ser and dimer-interface mutants based on the NLS mutant (NLSmut) construct of TDP-43 for
confocal microscopy imaging (Fig.?5). As known, all the mutants were mainly localized in the cytoplasm, and
the NLSmut mutant formed inclusions in few cells (Fig.?5A). The Cys mutations (C39/C50S) had very little
influence on inclusion formation, whereas the two dimer-interface mutants (L71/Y73A, V72/V74/Y43A) significantly
enhanced the formation of cytoplasmic inclusions (Fig.?5A,B). These results indicate that the N-terminal
dimerization is able to protect TDP-43 against formation of cytoplasmic inclusions.
The N-terminal dimerization of TDP-43 plays a role in its splicing activity. A well-known func
tion of TDP-43 is its ability to promote alternative splicing7, 50. To evaluate the role of NTD dimerization in
the splicing activity of TDP-43, we performed a CFTR exon 9 splicing assay20. We co-transfected the CFTR
mini-gene reporter and TDP-43 or its mutants into HEK 293 T cells and compared their splicing activities
(Fig.?6). As expected, overexpression of wild-type TDP-43 markedly enhanced CFTR exon 9 exclusion. However,
transfection of TDP-43-C39/C50S led to a decrease in CFTR exon 9 exclusion compared to wild-type
TDP43 (Fig.?6A,B) under the conditions of comparable nuclear localization (Fig.?6C) and similar expression levels
(Fig.?6D). Furthermore, when the dimer interface was disrupted by mutation of key residues (L71/Y73A or V72/
V74/Y43A), the CFTR exon 9 splicing activity was almost abolished, in contrast to the partial reduction of CFTR
exon 9 skipping activity caused by C39/C50S mutation (Fig.?6A,B). These results suggest that the dimer-interface
mutants lose their splicing activity due to impairment of the functional homodimer. Therefore, our findings
demonstrate that the N-terminal dimerization of TDP-43 is critical for its biological function.
TDP-43 is a nuclear factor that functions in promoting pre-mRNA splicing4. The C-terminal part has been
reported to have RNA binding ability and act as the source for aggregation. TDP-35, deletion of the N-terminal
NTD and NLS, may experience mislocalization to cytoplasm and cause formation of inclusions20. The present
study reveals that the N-terminal dimerization of TDP-43 is essential for its splicing activity. Zhang et al.
previously reported that the extreme N-terminal 10 residues are important for TDP-43 function in the nucleus46.
It is probably that deletion of the N-terminal 10 residues disrupts the structural ensemble of TDP-43 NTD and
consequently result in functional loss. Moreover, GFP fusion may alter the aggregation properties of TDP-43.
TDP-43 NTD is a well folded domain, and its solution structure has been elucidated by NMR under extreme
conditions47, 48. TDP(
) is not homogeneous in solution, so we used the GB1-fusion strategy to solve the
structure51. Biochemical and biophysical studies showed that TDP(
) remains a stable dimer under the
experimental conditions. Different from the structure solved in a dilute solution and presented as a monomeric state48, we
selected the intramolecular NOEs to calculate the monomeric structure from a stable dimeric state, analyzed the
dimer interface by mutagenesis and SEC, and constructed the dimer structure via computer modeling (Fig.?S7).
Nevertheless, the monomeric structure is overall similar to that from Laurents? group (PDB ID: 2N4P), despite
different protein samples and approaches being applied. Generally, TDP-43 NTD forms a ?-barrel structure
comprised mainly of two ?-sheets connected by a short ?-helix.
We found that a hydrophobic interface resides around the ?7-strand and plays a role in NTD dimerization.
With increased protein concentration, TDP-43 transforms to a tetramer through disulfide formation (Fig.?7).
Both the dimeric and tetrameric forms of full-length TDP-43 have splicing activity in nucleus (Fig.?6). The
formation of dimers or tetramers seems to protect TDP-43 against aggregation and inclusion formation in cytoplasm
(Fig.?5). Indeed, TDP-43 is a highly abundant protein localized in the nucleus2, 10, 50. Thus, we speculate that
TDP43 forms a dimer or tetramer under physiological conditions for pre-mRNA splicing in nucleus. However,
TDP35, a C-terminal fragment of TDP-43 without NTD and NLS, can induce its cytoplasmic localization, aggregation
or inclusion formation, and further loss of RNA processing activity20. In addition, TDP-43 can also form nuclear
or cytoplasmic granules under stress conditions, such as heat or arsenite treatment52, 53. Formation of the nuclear
granules might have relevance to the nuclear splicing activity of TDP-43. However, whether the N-terminal
dimerization of TDP-43 affects formation of nuclear stress granules remains future investigation.
The major finding of the present study is that the NTD fold of TDP-43 is essential for its dimerization and
consequent splicing activity, whereas the monomeric or aggregated forms may lose their biological functions.
This may provide mechanistic insights into the physiological function of TDP-43 and its related proteinopathies.
Materials and Methods
Construction of plasmids. The intrinsic Nde I site in the cDNA for human TDP-43 was previously mutated
by nonsense mutation. The cDNAs of TDP-43 fragments and their mutants were digested and ligated into pET22b
vector using Nde I/Xho I cloning sites. To prepare the GB1-fused C-terminal fragments, the cDNAs of TDP(
and its mutants were subcloned into a pET22b-GB1 plasmid. For eukaryotic expression, TDP-43 and its mutants
were constructed into pcDNA3.1/FLAG vector (Invitrogen) respectively. The Lys and Arg residues (K82, R83,
K84, K95, K97 and R98) in the NLS sequence of TDP-43 were mutated to Ala for cytoplasmic expression. All the
mutants were generated by site-directed mutagenesis and verified by DNA sequencing.
Protein expression and purification. All proteins were overexpressed in Escherichia coli BL21 (DE3)
strain (Invitrogen) and purified as described elsewhere29, 32. The His-tagged proteins were initially purified
through a Ni2+-NTA column (Roche), followed by SEC (Superdex-75 or Superdex-200, GE Healthcare). The
protein concentrations were determined spectrophotometrically using each extinction coefficient (?280).
Analytical size exclusion chromatography (SEC). All SEC experiments were performed on the AKTA
purifier facility with a fast protein liquid chromatography (FPLC) system (GE Healthcare) at room temperature.
Dynamic light scattering (DLS). The size and size distribution of proteins were determined by DLS on
a DynaPro instrument (Wyatt Technology Co., Santa Barbara) in disposable 100-?L micro cuvettes. The
proteins were diluted to different indicated concentrations with Buffer A. After centrifugation at 15,000g at 4 ?C for
30 min, the supernatants were subjected to DLS measurement. The scattering angle was fixed at 90? at a constant
temperature of 25 ?C. Data were collected for 30 scans and processed by Dynamics V7 software. The radius and
size distribution were calculated using the regularization algorithm provided by the software.
NMR spectroscopy and structure calculation. The C-terminal GB1 fusion was applied to improve the
homogeneity of the protein studied29, 51. To solve the solution structure of TDP(
)-GB1-C39/C50S by NMR,
the 15N/13C-labeled sample (~1 mM) was dissolved in Buffer B (20 mM phosphate, 50 mM NaCl, pH 6.5 and 8%
D2O) for data acquisition. All NMR data were acquired on a Bruker Avance 600-MHz spectrometer equipped
with a TCI CryoProbe (Bruker Biospin) at 25 ?C. The chemical-shift and NOE assignments, data processing, and
structure calculations were performed following the protocol for NMR structural analysis29.
Fourier transform infrared spectroscopy (FT-IR). FT-IR spectra were recorded on an ABB Bomem
MB-3000 spectrometer (Quebec, Canada) equipped with a deuterated triglycine sulfate detector. Data processing
was carried out as described elsewhere54. Second-derivative spectra were obtained using Savitsky-Golay
derivative function software for a 7-data-point window55. The second-derivative amide I spectra were analyzed to
estimate the relative secondary structure contents by manually computing the areas under the bands assigned to
a particular substructure.
Cell culture, transfection, Western blotting, and immunofluorescence microscopy. HEK 293
T cells were cultured in DMEM media (Invitrogen) supplemented with 10% fetal bovine serum (Gibco) and
penicillin-streptomycin under an ambience of 5% CO2 at 37 ?C. Transfection with plasmid was performed using
PolyJetTM reagent (SignaGen) according to the manufacturer?s instructions, and then the cells were harvested at
48 h after transfection. To examine the protein expression level, the transfected cells were lysed with a RIPA buffer
(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and cocktail protease inhibitor (Roche Applied
Science)) on ice for 30 min, and then centrifuged at 15,000 g at 4 ?C for 15 min. The lysates were mixed with the
2x loading buffer and subjected to Western blotting. The antibodies included mouse monoclonal anti-FLAG
(Sigma), mouse anti-TDP-43 (Abnova), mouse anti-GAPDH (Zen BioScience), and goat anti-mouse IgG-HRP
(Jackson Immuno-Research). The proteins were detected by an ECL detection kit (Thermo scientific). For
immunofluorescence microscopy, HEK 293 T cells were grown on glass cover slides for 48 h after transfection. The
adherent cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 1 h
and blocked with 5% BSA/10% FBS for 1 h. The fixed cells were then incubated with an antibody against FLAG
or TDP-43 for 1 h. After washing with PBS, the cells were labeled with an FITC-conjugated anti-mouse antibody
(Jackson Immuno-Research) for 1 h, and the nuclei were stained with Hoechst 33258 (Sigma) simultaneously. The
images were obtained using a Leica TCS SP4 confocal microscope (Leica Microsystems).
CFTR exon 9 splicing assay. The splicing assay was performed on the CFTR mini-gene reporter TG13T5
as described previously7, 20, 29, 50. Briefly, the reporter plasmid was co-transfected with TDP-43 or its mutants
into HEK 293 T cells. After 48 h, the cells were harvested and divided into two equal fractions. One was
subjected to Western blotting to examine the expression level, and the other was used to measure the CFTR exon 9
splicing activity. The total RNA was extracted with TRIzol reagent (Invitrogen) and the cDNA was generated by
retro-transcription using a ReverTra Ace-? kit (TOYOBO). PCR and agarose-gel electrophoresis were following
our previous protocols20. The intensity of each band was quantified by Scion Image software (Scion Corp). The
total intensity of exon 9+ and exon 9? of the vacant vector was set as a control, and the intensities of band exon
9? were compared with the control.
Coordinates and structure factors. The coordinates and structure factors have been deposited in the
Protein Data Bank with accession code 5X4F for TDP(
The authors thank Meng Wu and Jian Zhao for NMR data acquisition and analysis, and Wen-Tian He, Xiao-Fang
Yin and Hong-Wei Yue for cell culture and analysis. This work was supported by grants from the National Natural
Science Foundation of China (31670782, 31470758 and 31470786).
H.-Y.H. and L.-L.J. designed the research and wrote the manuscript; L.-L.J., W.X. and J.-Y.H. performed the
biochemical experiments; H.-Y.H. and W.X. carried out the NMR experiments and structural analysis; J.-T.Z. and
S.-N.Y. performed the FT-IR experiments and data analysis; and M.-J.L. and J.-H.H. contributed to the structural
Competing Interests: The authors declare that they have no competing interests.
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1. Ou , S. H. , Wu , F. , Harrich , D. , Garciamartinez , L. F. & Gaynor , R. B. Cloning and Characterization of a Novel Cellular Protein, Tdp43, That Binds to Human-Immunodeficiency-Virus Type-1 Tar DNA-Sequence Motifs . Journal of Virology69 , 3584 - 3596 ( 1995 ).
2. Ayala , Y. M. et al. Human, Drosophila, and C-elegans TDP43 : Nucleic acid binding properties and splicing regulatory function . Journal of Molecular Biology 348 , 575 - 588 , doi:10.1016/j.jmb. 2005 . 02 .038 ( 2005 ).
3. Buratti , E. & Baralle , F. E. Multiple roles of TDP-43 in gene expression, splicing regulation, and human disease . Frontiers in Bioscience-Landmark 13 , 867 - 878 , doi:10.2741/2727 ( 2008 ).
4. Buratti , E. & Baralle , F. E. The multiple roles of TDP-43 in pre-mRNA processing and gene expression regulation . Rna Biology 7 , 420 - 429 ( 2010 ).
5. Bhardwaj , A. , Myers , M. P. , Buratti , E. & Baralle , F. E. Characterizing TDP -43 interaction with its RNA targets . Nucleic Acids Research 41 , 5062 - 5074 , doi:10.1093/nar/gkt189 ( 2013 ).
6. Tollervey , J. R. et al. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43 . Nature Neuroscience 14 , 452 - U180 , doi:10.1038/nn.2778 ( 2011 ).
7. Buratti , E. & Baralle , F. E. Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9 . Journal of Biological Chemistry 276 , 36337 - 36343 , doi:10.1074/jbc. M104236200 ( 2001 ).
8. 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 . Journal of Biological Chemistry 280 , 37572 - 37584 , doi:10.1074/jbc. M505557200 ( 2005 ).
9. Buratti , E. & Baralle , F. E. TDP -43: gumming up neurons through protein-protein and protein-RNA interactions . Trends in Biochemical Sciences 37 , 237 - 247 , doi:10.1016/j.tibs. 2012 . 03 .003 ( 2012 ).
10. Winton , M. J. et al. Disturbance of nuclear and cytoplasmic TAR DNA-binding protein (TDP-43) induces disease-like redistribution, sequestration, and aggregate formation . Journal of Biological Chemistry 283 , 13302 - 13309 , doi:10.1074/jbc. M800342200 ( 2008 ).
11. Ayala , Y. M. et al. Structural determinants of the cellular localization and shuttling of TDP-43 . Journal of Cell Science 121 , 3778 - 3785 , doi:10.1242/jcs.038950 ( 2008 ).
12. Arai , T. et al. TDP -43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis . Biochemical and Biophysical Research Communications 351 , 602 - 611 , doi:10.1016/j.bbrc. 2006 . 10 .093 ( 2006 ).
13. Neumann , M. et al. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis . Science 314 , 130 - 133 , doi:10.1126/science.1134108 ( 2006 ).
14. Lagier-Tourenne , C. & Cleveland , D. W. Rethinking ALS : The FUS about TDP-43. Cell 136 , 1001 - 1004 , doi:10.1016/j. cell. 2009 . 03 .006 ( 2009 ).
15. Fang , Y. S. et al. Full-length TDP-43 forms toxic amyloid oligomers that are present in frontotemporal lobar dementia-TDP patients . Nature Communications 5 , doi:10.1038/Ncomms5824 ( 2014 ).
16. Lee , E. B. , Lee , V. M. Y. & Trojanowski , J. Q. Gains or losses: molecular mechanisms of TDP43-mediated neurodegeneration . Nature Reviews Neuroscience 13 , 38 - 50 , doi:10.1038/nrn3121 ( 2012 ).
17. Smethurst , P. , Sidle , K. C. L. & Hardy , J. Review: Prion-like mechanisms of transactive response DNA binding protein of 43 kDa (TDP43) in amyotrophic lateral sclerosis (ALS) . Neuropathology and Applied Neurobiology 41 , 578 - 597 , doi:10.1111/nan.12206 ( 2015 ).
18. Valle , C. & Carri , M. T. Which TDP -43 aggregates are toxic in ALS? Oncotarget 7 , 81973 - 81974 , doi:10.18632/oncotarget.13461 ( 2016 ).
19. Cascella , R. et al. Quantification of the Relative Contributions of Loss-of-function and Gain-of-function Mechanisms in TAR DNAbinding Protein 43 (TDP-43) Proteinopathies . Journal of Biological Chemistry 291 , 19437 - 19448 , doi:10.1074/jbc.M116. 737726 ( 2016 ).
20. Che , M. X. , Jiang , Y. J. , Xie , Y. Y. , Jiang , L. L. & Hu , H. Y. Aggregation of the 35-kDa fragment of TDP-43 causes formation of cytoplasmic inclusions and alteration of RNA processing . Faseb Journal 25 , 2344 - 2353 , doi:10.1096/fj.10- 174482 ( 2011 ).
21. Liu , Y. et al. A New Cellular Model of Pathological Tdp-43: The Neurotoxicity of Stably Expressed Ctf25 of Tdp-43 Depends on the Proteasome. Neuroscience 281 , 88 - 98 , doi:10.1016/j.neuroscience. 2014 . 09 .043 ( 2014 ).
22. Kitamura , A. et al. Interaction of RNA with a C-terminal fragment of the amyotrophic lateral sclerosis-associated TDP43 reduces cytotoxicity . Scientific Reports 6 , doi:10.1038/Srep19230 ( 2016 ).
23. Chen , A. K. H . et al. Induction of Amyloid Fibrils by the C-Terminal Fragments of TDP-43 in Amyotrophic Lateral Sclerosis . Journal of the American Chemical Society 132 , 1186 - + , doi:10.1021/ja9066207 ( 2010 ).
24. Medina , D. X. , Orr , M. E. & Oddo , S. Accumulation of C-terminal fragments of transactive response DNA-binding protein 43 leads to synaptic loss and cognitive deficits in human TDP-43 transgenic mice . Neurobiology of Aging 35 , 79 - 87 , doi:10.1016/j. neurobiolaging. 2013 . 07 .006 ( 2014 ).
25. Pesiridis , G. S. , Tripathy , K. , Tanik , S. , Trojanowski , J. Q. & Lee , V. M. Y. A ?Two-hit? Hypothesis for Inclusion Formation by Carboxyl-terminal Fragments of TDP-43 Protein Linked to RNA Depletion and Impaired Microtubule-dependent Transport . Journal of Biological Chemistry 286 , 18845 - 18855 , doi:10.1074/jbc.M111. 231118 ( 2011 ).
26. Yang , C. X. et al. The C-Terminal TDP -43 Fragments Have a High Aggregation Propensity and Harm Neurons by a DominantNegative Mechanism . Plos One 5 , doi:10.1371/journal.pone. 0015878 ( 2010 ).
27. Fuentealba , R. A. et al. Interaction with Polyglutamine Aggregates Reveals a Q/N-rich Domain in TDP-43 . Journal of Biological Chemistry 285 , 26304 - 26314 , doi:10.1074/jbc.M110. 125039 ( 2010 ).
28. Budini , M. et al. Cellular Model of TAR DNA-binding Protein 43 (TDP-43) Aggregation Based on Its C-terminal Gln/Asn-rich Region . Journal of Biological Chemistry 287 , 7512 - 7525 , doi:10.1074/jbc.M111. 288720 ( 2012 ).
29. Jiang , L. L. et al. Structural Transformation of the Amyloidogenic Core Region of TDP-43 Protein Initiates Its Aggregation and Cytoplasmic Inclusion . Journal of Biological Chemistry 288 , 19614 - 19624 , doi:10.1074/jbc.M113. 463828 ( 2013 ).
30. Mompean , M. et al. Structural characterization of the minimal segment of TDP-43 competent for aggregation? . Archives of Biochemistry and Biophysics 545 , 53 - 62 , doi:10.1016/j.abb. 2014 . 01 .007 ( 2014 ).
31. Mompean , M. et al. Structural Evidence of Amyloid Fibril Formation in the Putative Aggregation Domain of TDP-43. Journal of Physical Chemistry Letters 6 , 2608 - 2615 , doi:10.1021/acs.jpclett. 5b00918 ( 2015 ).
32. Jiang , L. L. et al. Two mutations G335D and Q343R within the amyloidogenic core region of TDP-43 influence its aggregation and inclusion formation . Scientific Reports 6 , doi:10.1038/Srep23928 ( 2016 ).
33. Sreedharan , J. et al. TDP -43 mutations in familial and sporadic amyotrophic lateral sclerosis . Science 319 , 1668 - 1672 , doi:10.1126/ science.1154584 ( 2008 ).
34. Pesiridis , G. S. , Lee , V. M. Y. & Trojanowski , J. Q. Mutations in TDP-43 link glycine-rich domain functions to amyotrophic lateral sclerosis . Human molecular genetics 18 , R156 - R162 , doi:10.1093/hmg/ddp303 ( 2009 ).
35. Arnold , E. S. et al. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43 . Proceedings of the National Academy of Sciences of the United States of America 110 , E736 - E745 , doi:10.1073/pnas.1222809110 ( 2013 ).
36. Lim , L. Z. , Wei , Y. Y. , Lu , Y. M. & Song , J. X. ALS-Causing Mutations Significantly Perturb the Self-Assembly and Interaction with Nucleic Acid of the Intrinsically Disordered Prion-Like Domain of TDP-43 . Plos Biology 14, doi:10.1371/journal.pbio. 1002338 ( 2016 ).
37. Guo , W. R. et al. An ALS-associated mutation affecting TDP-43 enhances protein aggregation, fibril formation and neurotoxicity . Nature Structural & Molecular Biology 18 , 822 - U102 , doi:10.1038/nsmb. 2053 ( 2011 ).
38. Alami , N. H. et al. Axonal Transport of TDP-43 mRNA Granules Is Impaired by ALS-Causing Mutations. Neuron 81 , 536 - 543 , doi:10.1016/j.neuron. 2013 . 12 .018 ( 2014 ).
39. Kuo , P. H. , Doudeva , L. G. , Wang , Y. T. , Shen , C. K. J. & Yuan , H. S. Structural insights into TDP-43 in nucleic-acid binding and domain interactions . Nucleic Acids Research 37 , 1799 - 1808 , doi:10.1093/nar/gkp013 ( 2009 ).
40. Johnson , B. S. et al. TDP-43 Is Intrinsically Aggregation-prone, and Amyotrophic Lateral Sclerosis-linked Mutations Accelerate Aggregation and Increase Toxicity . Journal of Biological Chemistry 284 , 20329 - 20339 , doi:10.1074/jbc.M109. 010264 ( 2009 ).
41. Zhang , Y. J. et al. Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity . Proceedings of the National Academy of Sciences of the United States of America 106 , 7607 - 7612 , doi:10.1073/pnas.0900688106 ( 2009 ).
42. Bozzo , F. et al. Structural insights into the multi-determinant aggregation of TDP-43 in motor neuron-like cells . Neurobiology of Disease 94 , 63 - 72 , doi:10.1016/j.nbd. 2016 . 06 .006 ( 2016 ).
43. Budini , M. , Romano , V. , Quadri , Z. , Buratti , E. & Baralle , F. E. TDP -43 loss of cellular function through aggregation requires additional structural determinants beyond its C-terminal Q/N prion-like domain . Human molecular genetics 24 , 9 - 20 , doi:10.1093/ hmg/ddu415 ( 2015 ).
44. Chang , C. K. et al. The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity . Biochemical and Biophysical Research Communications 425 , 219 - 224 , doi:10.1016/j.bbrc. 2012 . 07 .071 ( 2012 ).
45. Romano , V. , Quadri , Z. , Baralle , F. E. & Buratti , E. The structural integrity of TDP-43N-terminus is required for efficient aggregate entrapment and consequent loss of protein function . Prion 9 , 1 - 9 , doi:10.1080/19336896. 2015 . 1011885 ( 2015 ).
46. Zhang , Y. J. et al. The dual functions of the extreme N-terminus of TDP-43 in regulating its biological activity and inclusion formation . Human molecular genetics 22 , 3112 - 3122 , doi:10.1093/hmg/ddt166 ( 2013 ).
47. Qin , H. N. , Lim , L. Z. , Wei , Y. Y. & Song , J. X. TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA . Proceedings of the National Academy of Sciences of the United States of America 111 , 18619 - 18624 , doi:10.1073/pnas.1413994112 ( 2014 ).
48. Mompean , M. et al. The TDP-43 N-terminal domain structure at high resolution . Febs Journal 283 , 1242 - 1260 , doi:10.1111/ febs.13651 ( 2016 ).
49. Chang , C. K. & Huang , T. H. Untangling the structure of the TDP-43 N-terminal domain . Febs Journal 283 , 1239 - 1241 ( 2016 ).
50. Buratti , E. et al. Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping . Embo Journal 20 , 1774 - 1784 , doi:10.1093/emboj/20.7. 1774 ( 2001 ).
51. Bao , W. J. et al. Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization . Protein Expr Purif 47 , 599 - 606 ( 2006 ).
52. Bentmann , E. et al. Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43) . The Journal of biological chemistry 287 , 23079 - 23094 , doi:10.1074/jbc.M111. 328757 ( 2012 ).
53. McDonald , K. K. et al. TAR DNA-binding protein 43 (TDP-43) regulates stress granule dynamics via differential regulation of G3BP and TIA-1 . Human molecular genetics 20 , 1400 - 1410 , doi:10.1093/hmg/ddr021 ( 2011 ).
54. Yang , H. Y. , Yang , S. N. , Kong , J. L. , Dong , A. C. & Yu , S. N. Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy . Nature Protocols 10 , doi:10.1038/nprot. 2015 . 024 ( 2015 ).
55. Dong , A. & Caughey , W. S. Infrared methods for study of hemoglobin reactions and structures . Methods in enzymology 232 , 139 - 175 ( 1994 ).
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