From TDP-43/RNA complex formation to disease-linked TDP-43 aggregation through a structural and cellular approach

Domain: biology
Published: 2026-01-21
Editorial outcome: minor_revision

Abstract
Many RNA-binding proteins (RBP) have been associated to several neurodegenerative diseases for which RBP-rich cytoplasmic inclusions represent a major histological hallmark. However, among RBPs, the occurrence with which TDP-43, a nuclear mRNA-binding protein, is detected in cytoplasmic inclusions is exceptionally high. To unravel the underlying mechanisms, we focus our analysis on the structured N-terminal domain (NTD) of TDP-43, which is distinct among RBPs as this domain mostly initiates TDP-43 homotypic interactions. Through an in depth structural analysis, we successively show that the cooperative binding of TDP-43 along long GU-rich intronic sequences antagonizes NTD/NTD interactions between adjacent TDP-43 along mRNA. In contrast, the TDP-43 cooperativity facilitates NTD/NTD interactions between TDP-43 located on distinct GU-rich sequences. We hypothesize that NTD/NTD interactions between distinct GU-rich sequences efficiently allow the compaction of long introns in neurons under physiological conditions. However, when the binding of TDP-43 to RNA is discontinuous because of a lack of cooperativity, aberrant NTD/NTD interactions between adjacent TDP-43 take place, promoting the aggregation of TDP-43 RRMs (RNA Recognition Motifs) under stress conditions. Altogether, we provide a detailed view of the physiological assembly of TDP-43 on introns and the putative weaknesses of TDP-43 that makes it distinct in its propensity for aggregation compared to other RBPs.

Introduction
Many RNA-binding proteins (RBP) in the human genome harbor low complexity domains that initiate weak but numerous interactions, leading to the formation of subcellular compartments formed in the absence of membranes1. Under physiological conditions, RNA-related membraneless compartments that are mostly located in the nucleus, such as paraspeckles, speckles and nucleoli, fulfill many complex functions related to RNA transcription, mRNA splicing and ribosomal RNA processing2–4. However, in several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), nuclear RNA-binding proteins abnormally form cytoplasmic inclusions. Nuclear RBP aggregation in the cytoplasm potentially induces a loss of their nuclear function and/or a gain of toxic function in neurons5–8. Among the nuclear RBPs mislocalized in the cytoplasm, TDP-43 (encoded by TAR DNA Binding Protein, TARDBP gene) is the most frequently detected protein in cytoplasmic inclusions of ALS- and FTLD-affected neurons9–12. Since many of the TDP-43 pathological mutations are located in the C-terminal low complexity domain13 which is known for promoting phase separation, a transition from reversible TDP-43 liquid-like assemblies to an irreversible solid-like TDP-43 aggregation14–16 has been considered as the main factor of TDP-43 aggregation. However, pathological mutations in low complexity domains are not restricted to TDP-43 since they can be found in other RNA-binding proteins such as FUS17. The specificity of TDP-43 rather relies on its structured N-terminal domain (NTD) which is located at the N-terminal end (a.a. 4-77, Fig. 1A). TDP-43 NTD is known for its capacity to oligomerize18,19. Interestingly, besides NTD auto-assembly, no other protein has been identified as interacting directly with NTD or competing for the NTD-mediated multimerization. In addition, to our knowledge, no RBP other than TDP-43 itself harbors a similar NTD domain. The multimerization of TDP-43 NTD has been the subject of many studies to understand its potential role in the translocation of TDP-43 to the cytoplasm20,21, in TDP-43 aggregation20,22, and in its physiological role in splicing process18,23. Several reports indicated that NTD intermolecular interactions promote aberrant phase separation18,24. Especially, the formation of head-to-tail linear chains of NTD domain18 is considered as a major intermediate leading to TDP-43 aggregation in several models20. However, there is no structural data on the higher order assembly of TDP-43 when TDP-43 is complexed with long intronic GU-rich RNA sequences, its favorite nuclear RNA targets. The precise structural contribution of NTD/NTD interactions to the physiological role of TDP-43 in the processing of introns, as well as the interplay between TDP-43:RNA and NTD/NTD interactions in the structural transition from reversible RNA-rich TDP-43 assemblies to irreversible RNA-free TDP-43 aggregates, still remain to be dissected. Addressing these points may be critical to understand the successive steps leading to TDP-43 aggregation in neurodegenerative diseases.

Here, to tackle the above-mentioned issues, we first considered the higher order assembly of TDP-43 in the presence of GU-rich RNA. TDP-43 RRM1-2 binds cooperatively to GU-rich RNA based on an intermolecular interaction between RRM1 and RRM225. The cooperative binding of TDP-43 along RNA may either promote or prevent NTD/NTD interactions between adjacent TDP-43. Therefore, to document the interplay between the cooperative binding of TDP-43 to RNA and NTD/NTD interactions, we undertook a comprehensive structural characterization based on the analysis of extensive biochemical and NMR data using a long fragment of TDP-43 that contains both the NTD and the two RRMs. The structural analysis was complemented by thermodynamic data based on ITC analyses and large-scale views on the higher order assemblies of TDP-43 in complex with long oligonucleotides provided by Small Angle X-Ray Scattering (SAXS). We concluded that TDP-43 assemblies along GU repeats antagonize intermolecular NTD/NTD interactions between adjacent TDP-43 along RNA. On the other hand, long-range interactions between distant TDP-43:GU-rich RNA clusters can induce the compaction of long mRNAs. Accordingly, we then demonstrated that NTD-mediated interactions as well as the cooperative binding of TDP-43 to mRNA contribute to the formation of TDP-43 higher order assemblies in cells. Their contributions are interrelated since TDP-43 cooperative associations along GU-rich sequences favor long-range NTD/NTD interactions between distinct clusters and vice versa. To investigate TDP-43 aggregation, cells were exposed to mild oxidative stress in the presence of arsenite at low concentration. We evidenced a massive TDP-43 aggregation promoted by NTD/NTD interactions only when the cooperative association of TDP-43 to GU-rich mRNA was impaired. Therefore, in the absence of cooperative association, aberrant NTD/NTD interactions among TDP-43 adjacently bound to RNA promote TDP-43 aggregation. TDP-43 aggregation is due to inter-RRM irreversible assembly linked to the acetylation of some RRM lysine residues under oxidative stress conditions, as previously reported26. Interestingly, in agreement with the presented model, the expression of introns harboring long GU repeats in the nucleus significantly reduces the aggregation of wild type TDP-43 in the nucleus after arsenite stress.

Results
NTD/NTD interactions antagonize TDP-43/GU-rich cooperativity
In previous investigations at atomic level, we demonstrated that the TDP-43 tandem RRMs, RRM1-2 (a.a., 101-277), bind cooperatively to GU-rich RNA or GT-rich single-strand DNA repeats25. The cooperative binding occurs through an intermolecular interaction between the pocket around V220 in RRM2 of one monomer and the RRM1 loop 3 of an adjacent TDP-43 monomer when TDP-43 binds to GU-rich RNA or GT-rich ssDNA25. We then questioned the possible role of the structured NTD domain in the cooperative association of TDP-43 to RNA. At first sight, a link between two monomers via their respective NTDs may promote or secure the cooperative association (Hypothesis 1). However, conversely, the spatial arrangement resulting from the cooperative association may cause NTDs to be well separated from each other along the GU repeats. Therefore, the interaction between NTDs could be incompatible with a cooperative binding of TDP-43 to GU and GT repeats. In this case, NTD/NTD interactions may provide a negative contribution to the cooperative binding of TDP-43 to RNA or ssDNA (Hypothesis 2).

To challenge both hypotheses, we used a longer TDP-43 fragment (a.a., 1-277), including the full NTD domain, sufficiently soluble to be studied in vitro by using structural biology tools (ITC, NMR spectroscopy) (Fig. 1A, Supplementary Fig. 1 and 2A). We first verified that the presence of the NTD domain does not induce major structural changes or new interactions with RRM1-2. For this, we compared the chemical shifts of the RRM1-2 residues when the fragment contains or lacks the NTD domain (a.a., 1-277 vs a.a., 101-277, Fig. 1B and Supplementary Fig. 2A, B). We also constructed two NTD mutants, R52E and E17R, which significantly impair the NTD/NTD multimerization on opposite sides of the NTD domain and display the molecular weight of a protein monomer (Supplementary Fig. 1). In the absence of RNA or ssDNA, the peaks of RRM1-2 residues are superimposable for all the fragments tested (NMR spectra in Fig. 1B and Supplementary Fig. 2B). In addition, we reconstituted the NTD/NTD interface with an equimolar protein mixture of 15N-labelled R52E and unlabeled E17R (R52E + E17R, a.a., 1-277). The reconstitution of NTD/NTD interactions involves only the non-mutated face of each complementary mutants E17R and R52E. In this case, the peaks of NTD residues involved in NTD/NTD multimerization disappear (as compared to respective residues in NTD mutant alone), attesting to the recovery of the NTD/NTD interface (Fig. 1B). Each mutant contributes to form the dimer interface through its non-mutated side as confirmed from NMR experiments by using 15N-labelled R52E or 15N-labelled E17R (Supplementary Fig. 3). However, no residue or group of residues displays major chemical shifts in RRM1-2 due to NTD/NTD interactions (Fig. 1B and Supplementary Fig. 2B).

Next, we probed whether the cooperative association of TDP-43 to GT repeats depends on NTD/NTD interactions by using ITC analysis. We first tested a stretch of 6 GT repeats, (GT)6, which can only accept one TDP-43 unit. In this case, the presence of the NTD, whether functional (WT) or not (NTD mutant, E17R), does not affect the stoichiometry nor the affinity of TDP-43 for (GT)6 (Fig. 1C, Supplementary Fig. 4A).

In the presence of (GT)12 (12 GT repeats), two RRM1-2 (a.a., 101–277) can bind to the same oligonucleotide (Fig. 1D and Supplementary Fig. 4B; RNA/protein molar ratio, N~0.5), which lead to the presence of 2 plateaus, a major feature of cooperative association to (GT)12. The calculations confirmed the strong cooperativity of isolated RRM1-2 alone since the value of the ratio, KD1/KD2 is very high, meaning that the binding affinity of the second monomer to (GT)12 is stronger than the first one. However, when we used the long fragment which contains both RRM1-2 and NTD (a.a., 1-277), the cooperative phenomenon is strongly disrupted (low KD1/KD2) suggesting that NTD/NTD interactions are an obstacle for the cooperativity (red plot in Fig. 1D). To confirm this point, we also performed ITC experiments by using the R52E mutant, which prevents the intermolecular association between NTDs. The ITC profile shows that the cooperative association of TDP-43 R52E mutant to (GT)12 oligonucleotide is then restored (blue plot in Fig. 1D). TDP-43 E17R mutant displays similar behavior as R52E mutant (yellow plot in Fig. 1D). Importantly, when we generated NTD/NTD interactions with an equimolar mixture of R52E and E17R mutants (reconstituted dimer, R52E + E17R, a.a., 1-277), the cooperative assembly of TDP-43 to (GT)12 once again significantly decreases (green plot in Fig. 1D, Supplementary Fig. 4B). Moreover, TDP-43 targets specifically GT/GU repeats. Accordingly, TDP-43 displays no affinity for poly (A) sequence as showed by using EMSA (Supplementary Fig. 5A) and very limited chemical shifts in the presence of poly (A) sequence or CA repeats in NMR spectra (Supplementary Fig. 5B). In this case, the introduction of a stretch of poly-A (A12) between two (GT)6 repeats, interrupting the possibility of continuous binding of R52E to GT-rich ssDNA, prevents the cooperative association of R52E (Supplementary Fig. 4C). Together, the results clearly confirm the negative contribution of NTD/NTD interactions to the cooperative binding of TDP-43 to long (GT)12 repeats.

NTD does not interfere with RRM1-2 binding to nucleic acids
To further document the interplay between the cooperative binding to nucleic acids and NTD/NTD interactions, we performed an extensive NMR analysis of wild type TDP-43 and R52E in the presence of 6 or 12 GU/GT repeats. By comparing the residues involved in RNA interactions in the wild type RRM1-2 (a.a., 101-277), wild type TDP-43 (a.a., 1-277), and TDP-43 R52E mutant (a.a., 1-277), we found that NTD/NTD interactions did not significantly interfere with the chemical shifts of RNA-binding residues in RRM1-2 domains in the presence of short (GT)6 oligonucleotide (Supplementary Fig. 6 and 7). We can therefore conclude that NTD/NTD interactions have no impact on the binding of RRM1-2 to short nucleic acids. To confirm this point, we also used an equimolar mixture of 15N-labelled R52E and unlabeled E17R mutants (a.a., 1-277) to reconstitute a R52E/E17R dimer, as demonstrated by the disappearance of the resonance peaks of NTD-associated residues of 15N-labelled R52E protein. In the presence of either (GT)6 or (GT)12 oligonucleotide, the chemical shifts for RRM1-2 residues involved in the binding with nucleic acids are similar for all the protein forms tested (Supplementary Fig. 6 and 7). Similar observations were made with GU-rich RNA repeats (Supplementary Fig. 8, see arrows). We can therefore conclude that NTD/NTD interactions mostly affect the cooperative binding but not the interaction of RRM residues with nucleic acids.

Cooperativity weakens the NTD/NTD interface
We then focused on whether the residues associated with NTD/NTD interactions are affected by the binding to GT or GU repeats. NTD/NTD interactions lead to significant peak broadening of NTD residues, which affects their detection in the NMR spectra. A similar broadening was observed when the 15N-labelled TDP-43 R52E mutant interacts with an unlabeled TDP-43 E17R mutant at equimolar concentration (Supplementary Fig. 9). The presence or absence of (GT)6 or (GT)12 did not significantly change this trend, which indicates that NTD/NTD interactions are preserved (Supplementary Fig. 10). However, an in-depth analysis of the chemical shifts and the heights of the resonance peaks revealed significant differences (Fig. 2A, B). Several NTD resonance peaks of wild type 15N-labelled TDP-43 as well as of the equimolar mixture of 15N-labelled TDP-43 R52E and unlabeled TDP-43 E17R reappear in the presence of (GT)12 more markedly compared to (GT)6 (Fig. 2B, residues: I18, S29, V31, T32, V74; Supplementary Fig. 9). Likewise, several NTD peaks also appear when wild type TDP-43 (a.a., 1-277) interacts with (GU)12 RNA compared to (GU)6 RNA (Supplementary Fig. 8, residues: I18, S29, T32, G37). Importantly, we observed larger chemical shift perturbations for resonances of several NTD residues located at the NTD/NTD interface in the presence of (GT)12 compared to in the presence of (GT)6 (E17, R55 and G53, Supplementary Fig. 11, Fig. 2A, B). In contrast, the resonance peaks of R52E NTD residues, unable to initiate NTD/NTD interactions, are always observable with no differences among the chemical shifts under all conditions. The chemical shifts of resonance peaks of NTD residues located at the NTD/NTD interface may indicate reduced NTD/NTD interactions in the presence of (GT)12 or (GU)12 compared to (GT)6 or (GU)6.

To more clearly probe NTD/NTD interactions, we performed competition assays using a truncated and unlabeled E17R NTD fragment (a.a., 1-93, lacking the RRM1-2 domains). E17R NTD does not bind to RNA but interacts with the NTD of R52E mutant (a.a 1-277) (Supplementary Figs. 10 and 12). Upon increasing concentrations of an unlabeled E17R NTD, the peaks of 15N-labelled NTD residues related to the NTD/NTD interface become increasingly apparent and undergo large chemical shifts18. This phenomenon is due to the dynamical exchange between NTDs that enables the reappearance of the peaks of NTD residues. The lower the affinity between NTDs, the more pronounced the reappearance of NTD peaks should be. Here, we used a different version of this assay18 (Fig. 2C, D). We added unlabeled E17R NTD (a.a., 1-93) in excess to compete the NTD/NTD interface involving 15N-labelled TDP-43 R52E that initially interacts with an unlabeled TDP-43 E17R in the presence of (GT)12 or (GT)6. Interestingly, we observed the reappearance of NTD shifted peaks related to 15N-labelled R52E with (GT)12 and more significantly than with (GT)6 (Supplementary Fig. 12). This probably means that the E17R NTD can access to the TDP-43 NTD interface more easily in presence of (GT)12 than with (GT)6. Furthermore, we then used a long oligonucleotide, (GT)6 A12 (GT)6, in which a linker of 12 adenine nucleotides was added to prevent the cooperative association between adjacent TDP-43 along (GT)6 A12 (GT)6, in agreement with ITC analysis (Supplementary Fig. 4C). In contrast with (GT)12, (GT)6 A12 (GT)6 does not enable the reappearance of TDP-43 NTD resonance peaks upon the addition of unlabeled E17R NTD fragment (a.a., 1-93) (Fig. 2C, D). The discontinuous binding of TDP-43 to (GT)6 A12 (GT)6 thus secures NTD/NTD interactions.

In summary, NTD/NTD interactions between adjacent TDP-43 proteins are limited when TDP-43 binds cooperatively to long (GT)12 repeats, unlike non-cooperative association to short or longer nucleic acids.

NTD/NTD interactions compact long nucleic acids
To investigate structural changes on a larger scale than accessible by NMR spectroscopy, we performed SAXS analysis of in vitro-purified TDP-43:oligonucleotide complexes. We used four different protein combinations (a.a., 1-277) to explore the putative role of NTD/NTD interactions: the wild type TDP-43 and mutants R52E, E17R alone or together. The acquisition of the SAXS images was coupled to a Size Exclusion Chromatography (SEC) column (Supplementary Fig. 13A). The SEC chromatograms and the SAXS images were recorded during the elution. In the presence of (GT)12 for which the binding is cooperative (Fig. 1D), we observed that single mutants (blue and yellow trace) display similar elution volumes as WT (red trace) (Supplementary Fig. 13A). However, NTD/NTD interactions-deficient mutants display lower elution volumes due to a less compacted conformation than wild type TDP-43 in the presence of (GT)6 A12 (GT)6, which cannot allow a continuous binding of TDP-43 because of the stretch of A12 present in the middle of the oligonucleotide.

The SEC-SAXS data allowed us to estimate the radius of gyration (Rg) and the maximum distance (Dmax) of the nucleoprotein complexes (Fig. 3, Supplementary Figs. 14 and 18). The results confirmed the observations based on the elution volumes. NTD/NTD interactions do not induce the compaction of nucleoprotein when TDP-43 is cooperatively associated to (GT)12. Indeed, when (GT)12 was used, the sizes of the nucleoprotein complexes are nearly identical for all the TDP-43 constructs, for wild type TDP-43 (162 Å, maximum diameter) alongside the range observed for the mutants (159 Å–165 Å), and therefore independent on the NTD/NTD interactions (Fig. 3A, Supplementary Fig. 13B, C and 15A). In addition, full atomic models of two TDP-43 bound to (GU)12 were generated by using the DADIMODO web server (https://dadimodo.synchrotron-soleil.fr)27. These models were obtained by using a constructed model based on available 3D structure of NTD domain and a model of TDP-43 dimer from our previous investigations25 and SAXS curves. In all the models, the two NTD domains are positioned far apart, which should limit NTD/NTD interactions. Here, the obtained models for the wild type as well as R52E mutant (a.a., 1-277) fit well the experimental SAXS curves with low discrepancy values (χ2) around 1.35–1.39 and 1.61–1.64, respectively (Supplementary Fig. 16).

To investigate the putative existence of a closed form displaying NTD/NTD interface between adjacent TDP-43 along (GU)12, we applied distance constraints (4–5 Å) between R52 residue from NTD of one TDP-43 unit, and E17 residue from NTD of the second TDP-43 unit. Interestingly, the best-fitting models display significantly elevated χ2 values, 3.12 or 3.60 when R52 (from the first unit) interacts with E17 (from the second unit) or vice versa (E17/R52), respectively (Supplementary Fig. 17). Then, the best-constrained conformation was subsequently used as the starting point for a new calculation performed without any distance constraints by using DADIMODO program. Strikingly, the result of this calculation indicates that the best conformation obtained, with a χ2 value of 1.33 similar to the initial calculations, corresponds to an open model in which the two NTD domains are well separated by approximately 72 Å (Supplementary Fig. 17). In conclusion, all of the best generated solutions displayed the models of TDP-43 dimer cooperatively associated to (GU)12 where the two adjacent NTDs are separated. Moreover, the closed conformation (harboring the NTD/NTD interface) gives a worse fit than the open state.

In contrast, in the case of (GT)6 A12 (GT)6 oligonucleotide, with which the cooperative association of TDP-43 is not possible, we observed smaller Dmax values, indicating compaction of the nucleoprotein complexes when NTD/NTD interactions occur. This behavior is observed only for wild type TDP-43 (Dmax: 166 Å) and for an equimolar mixture of E17R and R52E (Dmax: 168 Å) (Fig. 3B and Supplementary Fig. 13B, C and 15A). In addition, the complexes formed in the presence of (GT)6 A12 (GT)6 remain extended with R52E (Dmax: 203 Å) or E17R (Dmax: 200 Å), when they are used alone. Similar results were obtained in the presence of GU RNA repeats oligonucleotides (Supplementary Fig. 14A, B and 15B). Therefore, as long as TDP-43 proteins are not positioned adjacently along the long GT- or GU-rich nucleotides through a cooperative association, TDP-43 NTD/NTD interactions could promote intermolecular bonds to compact nucleoprotein complexes. To further probe this model, the number of GT repeats was progressively increased from 9 to 24 (Fig. 3C, Supplementary Figs. 18 and 19). We then measured the evolution of the Dmax in the presence of WT or R52E. We noticed a steady increase in Dmax versus the number of GT repeats, in absence of NTD/NTD interactions (R52E mutant). In contrast, when NTD/NTD interactions are possible, we observed no further increase in Dmax for either wild type TDP-43 or a mix of R52E and E17R, in the presence of the longest oligonucleotides (GT)24. These results indicate that very long GT repeats complexes can bend on themselves because of long-ranged NTD/NTD interactions between non-adjacent TDP-43 (Fig. 3C).

Because of limited NTD/NTD interactions among adjacent TDP-43 proteins, the availability of NTD for interactions between NTDs located on distant clusters of GT repeats can be strongly promoted. NTD/NTD interactions and cooperativity would then be compatible to mutually reinforce instead of opposing each other. To document this point, we performed protein cross-linking assays in the presence of (GT)12 as control and (GT)12 A6 (GT)12 A6 (GT)12, the latter corresponds to 3 distinct GT clusters separated by 6-nt long adenine linkers (Fig. 3D). Consistently with the NTD-mediated compaction, cross-linking assays reveals the formation of tetramers of wild type TDP-43 complexed with (GT)12 A6 (GT)12 A6 (GT)12 that are not present with R52E or with (GT)12 ssDNA. The presence of tetramers most likely results from long-ranged NTD/NTD interactions that promote the formation of molecular bridges between two GT-rich clusters, each containing two units of TDP-43.

NTD/NTD interactions and cooperativity interplay in cells
To assess the nature of TDP-43-associated intermolecular interactions in a cellular context, we used MT bench assays. In this assay, microtubules are used as intracellular platform to detect RNA:RBP interactions in a cellular context but also the mixing:demixing between RBPs in mRNA-rich compartments that are formed following the expression of two different RBPs. We have previously taken advantage of this approach to reveal TDP-43 residues that are critical for its cooperative association with mRNA but also to analyze TDP-43 homotypic interactions28. Briefly, two different RBPs labeled with two different fluorophores (RFP and GFP) are brought onto microtubules through a fusion with a microtubule-binding domain (MBD). In each compartment, the enrichments of RFP- and GFP-labeled proteins are then measured. The measurement of R², the degree of linear correlation between the enrichment of the two RBPs in the compartments provides a mixing score between two RBPs (Fig. 4A, B). The mixing is low (R²«1) when proteins form distinct RFP- or GFP-labeled compartments. The automatic acquisition of images by a high-resolution HCS imager allows obtaining robust mixing scores in 96-well plates over a large number of cells via an entirely automated pipeline (Fig. 4A, Supplementary Fig. 21A and Supplementary Fig. 21B for experimental replicates).

To probe the self-assembly of TDP-43 using MT bench assays, we prepared a combination of single- or double-point mutations. As previously demonstrated, G146A prevents the cooperative binding of TDP-43 to mRNA, but G146A does not alter TDP-43 structure nor TDP-43 affinity for short RNAs, according to NMR and ITC analyses25. R52E and E17R are two mutations preventing NTD/NTD interactions that we already used and validated (Fig. 1D). However, the co-expression of these two mutants allow the reconstitution of NTD/NTD interactions leading to form a dimer involving the non-mutated face of each complementary mutants E17R and R52E. The mutated face is ineligible to form NTD/NTD interactions.

R52E mutation is sufficient to impair the mixing with wild type TDP-43 (Fig. 4C) because each wild type unit possesses two complementary NTD/NTD interfaces and can form a multimer. However, E17R or R52E NTD mutants respectively preserve only one NTD/NTD interface, which is complementary to one of the wild type NTD faces. As expected, R52E mixes well with E17R to form a dimer. R52E displays a significant demixing with wild type (-15.3%) since R52E interacts with wild type NTD through only one binding site in contrast with wild type NTD.

Furthermore, as expected, the double mutant, E17R-R52E, markedly reduces the mixing with wild type TDP-43 because the double mutation impairs NTD/NTD interactions. Interestingly, the double mutation G146A-R52E further decreases the mixing with wild type TDP-43 compared to G146A or R52E alone (-26.3% versus -11.2% and -13.0%, respectively, Fig. 4D). Therefore, cooperativity and NTD/NTD interactions contribute incrementally to the mixing with wild type TDP-43 in compartments. Accordingly, the mixing of RFP-labeled G146A-R52E increases when GFP-labeled TDP-43 harbors either R52E or G146A mutation to reduce its capacity for self-assembly through NTD/NTD interactions or cooperativity, respectively (Fig. 4D, E).

Next, we explored the interplay between the cooperative binding of TDP-43 to mRNA and NTD/NTD interactions. To this end, we compared changes in mixing scores among cooperativity-deficient RFP or GFP-labelled G146A mutants (Fig. 4E, lower panel). We found that the double mutant G146A-R52E induces a strong demixing with G146A (-12.4%) comparable to cooperative-proficient mutant. These results indicate that NTD/NTD interactions occur in the absence of cooperativity. In contrast, among NTD-deficient RFP or GFP-labelled R52E, G146A mutation induces a significant but moderate demixing (-4%). Therefore, cooperativity occurs in the absence of NTD/NTD interactions, though but to a less extent than for wild type TDP-43 (Fig. 4E, upper panel).

Based on all these results, we propose a scheme for TDP-43 higher order assemblies consistent with the results obtained from MT bench assay (interplay between cooperativity and NTD/NTD interactions) and SAXS approach (NTD/NTD interactions are repressed for adjacent TDP-43) (Fig. 4F). We consider a cluster as multiple TDP-43 proteins bound along the same GU-repeats. From SAXS data, we know that NTD is available for inter- but not intra-cluster interactions. Therefore, NTD/NTD interactions should promote the recruitment of several TDP-43 from two distinct clusters to initiate multiple NTD/NTD interactions (Fig. 4F, upper panel). In turn, cooperativity may secure the formation of clusters (Fig. 4F, lower panel).

In summary, the analyses of the data obtained with the MT bench assay unravel that NTD/NTD interactions and TDP-43 cooperative association with mRNA each contribute to promote the formation of NTD-linked TDP-43 clusters.

Cooperativity limits NTD-mediated RRM aggregation in cells
In one of the TDP-43 aggregation models, TDP-43-rich inclusions in cells appear following inter RRM interactions26. More precisely, under stress conditions, such as exposure to arsenite, acetylation of lysine residues leads to aggregation between RRMs26. Accordingly, in HeLa cells expressing HA-tagged TDP-43, we observed that even low concentrations of arsenite (80 µM for 1 h, Fig. 5A) are able to generate the formation of mRNA-free of detergent-insoluble TDP-43 aggregates (see reference 25). Importantly, under the same conditions, when other RBP harboring RRM domains such as HuR and FUS are expressed in cells in a similar manner to TDP-43, we did not observe any significant nuclear RBP aggregation as in the case of TDP-43 (Fig. 5B). These results demonstrate the specific sensitivity of TDP-43 to oxidative stress. Next, using TDP-43 mutants that we have already characterized in vitro and with MT bench assays, we measured the formation of nuclear aggregates in cells expressing different mutants at similar levels to make a fair comparison (Supplementary Fig. 22B). When TDP-43 does not bind cooperatively to RNA (G146A mutant) very low arsenite concentration promotes a massive aggregation (down to 5 µM for 1 h, Fig. 5A) without sequestering mRNA, as expected for an RNA-free TDP-43 aggregation. The aggregation level is much more marked than with wild type TDP-43 (Fig. 5A). Interestingly, without NTD/NTD interactions, R52E mutation totally suppresses TDP-43 aggregation induced by arsenite (Fig. 5C). The double mutation, R52E-G146A, also completely suppresses the massive aggregation of G146A (Fig. 5C). Therefore, cooperativity antagonizes TDP-43 aggregation mediated by arsenite. In contrast, interactions between NTDs strongly promote the formation of TDP-43 aggregates. To further support these results, we analyzed the extraction of nuclear TDP-43 from cells treated with arsenite after short exposure to detergent (Supplementary Fig. 23). R52E-G146A and R52E mutants are better extracted than wild type TDP-43 and G146A, as expected if NTD/NTD interactions reduce TDP-43 solubility after arsenite treatment. In addition, we tested whether the nuclear TDP-43 aggregates are truly irreversible assemblies by washing out arsenite for 90 min (Supplementary Fig. 24). As control, stress granules which are known RBP-rich reversible condensates29 mostly disappear after arsenite removal. In contrast, the presence of wild type and G146A TDP-43 puncta in the nucleus persists, consistent with TDP-43 aggregation (or the formation of solid-like condensates with very low dissociation kinetics).

At the light of these results, we therefore performed a series of experiments to test the role of lysine acetylation within RRM1-2 in the aggregation of TDP-43. Two pairs of lysine residues, known to undergo acetylation following arsenite stress, were mutated to glutamine and arginine26. The glutamine substitution mimics the acetylation while the arginine substitution abolishes the ability to receive the acetylation modification. The double mutation, K136R-K137R, does not induce any noticeable consequence regarding the aggregation of TDP-43. On the other hand, the double mutation, K145R-K192R, prevents the aggregation of TDP-43 after arsenite exposure, even with the mutation G146A (Fig. 6A, B), while the double mutant, K145Q-K192Q, promotes the aggregation of TDP-43 (Supplementary Fig. 25A). Therefore, the absence of acetylation of these two residues simultaneously seems important because mutation of only one of these lysine residues is not sufficient to prevent the formation of aggregates (Supplementary Fig. 25B).

In view of the results of the arsenite-mediated aggregation assays, we propose the following mechanism of TDP-43 aggregation. When TDP-43 binds to mRNA in a non-cooperative manner, interactions between TDP-43 NTDs along RNA are then possible, as observed with cross-linking assays (Fig. 3D). Consequently, under stress conditions, there are still NTD/NTD interactions between nearby TDP-43 even after their release from mRNA. This NTD/NTD link between RNA-free TDP-43 increases the occurrence of interactions between acetylated RRMs to promote aggregation. Long-ranged NTD/NTD links between distinct TDP-43 clusters under physiological conditions are less likely to increase the occurrence of RRM intermolecular interactions.

To further challenge this hypothesis, we used a well-characterized splicing reporter in which the presence of an intronic 13 GU repeats is recognized by TDP-43 to control the excision of exon 9 in the CFTR gene30–32. This construct has been previously validated since the expression of TDP-43 is necessary to control the splicing of CFTR exon 9. We then modified this splicing reporter by adding a 7 nt-long adenine nucleotide linker right in the middle of the poly(GU) sequence. In this case, the cooperative binding of TDP-43 is no longer possible. Consistently, the splicing of CFTR exon 9, controlled by TDP-43, is impaired (Fig. 6C, Supplementary Fig. 26). After these different controls, we measured the aggregation of TDP-43 as a function of the expression of splicing reporter pre-mRNA (GFP expression level). The aggregation of TDP-43 in the nucleus following exposure to arsenite continuously decreases with the expression level of the pre-mRNA containing 13 GU repeats (Fig. 6C). Conversely, increasing the expression of pre-mRNA containing (GU)6 A7 (GU)7 does not limit nuclear TDP-43 aggregation. In summary, the results indicate that the cooperative association of TDP-43 prevents NTD/NTD interactions between adjacent TDP-43 to drastically decrease nuclear TDP-43 aggregation under stress conditions.

Methods
Protein production and purification
The recombinant His6-tagged NTD fragment, His6-tagged RRM1-2 fragment and His6-tagged NTD-RRM1-2 fragment of human TDP-43 were carried by pET plasmids (Supplementary Table 1).

Point mutations were introduced in pTDP-NTD_1-93 and pTDP-NTD-RRM1-2_1-277 expression plasmids by the ‘Quikchange II XL site-directed mutagenesis kit’ from Stratagene and corresponding oligonucleotides (Eurofins Genomics). DNA sequencing (Eurofins Genomics) checked all mutant sequences.

Proteins were overexpressed in E. coli strain BL21 (DE3). Cells grew at 37 °C in 2YT-kanamycin medium for unlabeled proteins or in minimal medium M9 supplemented with 15NH4Cl for 15N-labeled proteins. IPTG, which need to be a final concentration of 1 mM, was added into the culture when the optical density of the culture reached 0.8. IPTG is a reagent for efficiently initiating the transcription of lac operon to express the target gene in the operon. The bacteria grew continually for about 3.5 h.

Bacteria were gained and washed with 25 mM Tris-HCl buffer (washing buffer) at pH 7.6, containing 0.5 mM DTT and 2 M KCl. The pellet was suspended with about 20 mL-35 mL of 25 mM Tris-HCl buffer (lysis buffer) at pH 7.6, containing 0.5 mM DTT, 1 mM PMSF, EDTA-free protease inhibitor Cocktail (Roche) and 2 M KCl, and broken by sonication on ice (Bioblock Vibracells sonicator, model 72412). The final mixture was centrifuged for 30 min at 74,000 × g 4 °C in a TL100 Beckman centrifuge. The supernatant was collected for protein purification.

The collected supernatant was purified following the manufacturer’s recommendations (Qiagen). 7.5 mM imidazole was added into the collected supernatant and the supernatant was incubated overnight at room temperature with Ni2+-NTA agarose (Qiagen) (20 mg of proteins/mL of resin) which has already pre-equilibrated in washing buffer. After incubation, the protein can bind to the resin via Histidine to form the polymer. The polymer was transferred to an Econo-Pac chromatography column (Bio-Rad) and then washed extensively with lysis buffer containing 10 mM imidazole. After that, the polymer was washed with washing buffer containing 15 mM imidazole. Then, the elution of the protein was obtained by increasing the concentration of imidazole from 20 mM to 250 mM, in 25 mM Tris-HCl buffer (buffer A) at pH 7.6, containing 0.5 mM DTT and 500 mM KCl. Thermo Scientific™ NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer detected the concentration of the elution of protein. The fractions (usually from 50 mM to 250 mM imidazole) of pure protein were pooled and incubated with the target protein. The His6-tag peptides were removed from the target protein by incubating with the target protein (0.5 μM TEV to ∼30 μM protein) in buffer A, containing 1 mM DTT and 1 mM EDTA. The digestion was overnight at room temperature. A PD-10 column (GE Healthcare) was used for imidazole removing and buffer exchange. The TEV protease and His6-tag peptides from the target protein were caught on the Ni2+-NTA agarose column, and the target protein was collected in the pass-through fractions. The purity of the protein was verified by SDS-PAGE to verify that the desired TDP-43 fragments were finally obtained. To verify the NTD-RRM1-2 fragment of TDP-43, 12% resolving gel was used, while 15% resolving gel was used for the RRM1-2 fragment and the NTD fragment verification.

Purified proteins were stored in 25 mM Tris-HCl buffer at pH 7.6, containing 1 M KCl and 0.5 mM DTT, at -80 °C.

Isothermal titration calorimetry measurements
ITC experiments were performed at 25 °C by a MicroCal PEAQ-ITC isothermal titration calorimeter (Malvern Instruments). All the samples were prepared in 20 mM Hepes buffer at pH 7.6, containing 100 mM KCl and 2 mM TCEP. The protein concentration in the micro-calorimeter cell (0.2 mL) varied from 13 μM to 18 μM. 37 injections of 1 μL (or 39 injections of 1 μL) of oligonucleotide solution [(GT)6 at concentration 200 μM, (GT)12 at concentration 100 μM and (GT)6 A12 (GT)6 at concentration 100 μM] were carried out at 90 s intervals, with stirring at 750 rpm. Theoretical titration curves were fitted to the experimental data using the MicroCal PEAQ-ITC Analysis Software provided by the manufacturer. This software models the relationship between the heat released during each injection and several parameters: ΔH (enthalpy change, in kcal·mol⁻¹), K (dissociation constant, in M), n (number of binding sites per monomer), as well as the total protein concentration and the concentration of free and total oligonucleotide. Changes in the free energy and entropy upon binding were calculated from equilibrium parameters obtained.

Samples in the presence of (GT)6 and (GT)6 A12 (GT)6 were fitted with the one set of sites model, which enables the determination of the association constant K, the binding enthalpy ΔH, and the stoichiometry n of the interaction. The cumulative heat Q after each injection is given by Eq. (1):

Q=M0 V0 [nΔH KX / 1+KX] (1)

where V0 is the cell volume, M0 is the total protein concentration, and X is the free oligonucleotide concentration in the active volume.

Samples in the presence of (GT)12 were fitted with the two sets of sites model, enabling determination of the association constants (K1, K2), binding enthalpies (ΔH1, ΔH2), and site stoichiometries (n1, n2) for each binding site. The cumulative heat Q after each injection is described by Eq. (2):

Q=M0 V0 [n1 ΔH1 K1 X / 1+K1 X + n2 ΔH2 K2 X / 1+K2 X] (2)

where V0 is the cell volume, M0 is the total protein concentration, and X is the free oligonucleotide concentration in the active volume.

For both models, the heat observed for each injection, qi, is calculated as shown below in Eq. (3):

qi=ΔQi=Qi + dV/V0 [Qi + Qi-1]/2 - Qi-1 (3)

This approach follows respectively, the one set of sites and two sets of sites models formalism implemented in PEAQ-ITC and is based on established ITC methodology51,52.

Nuclear magnetic resonance of TDP-43 fragments
For NMR samples preparation, 30 μM of purified proteins (15N-labeled NTD-RRM1-2 fragments, NTD fragments or RRM1-2 fragments) were prepared as free protein samples. For DNA oligonucleotide-bound protein samples, (GT)-rich DNA oligonucleotides [(GT)6: 35 μM, (GT)6 A12 (GT)6: 17.5 μM or (GT)12: 17.5 μM] were heated at 95 °C for 1 min and incubated with 30 μM purified proteins. The same treatments were done for 15N-labeled NTD-RRM1-2 fragments with (GU)-rich RNA oligonucleotides [(GU)6, (GU)6 A12 (GU)6 or (GU)12] to prepare RNA-bound protein samples.

In dimer reconstruction experiments, the mixture of proteins contained 30 μM 1-277_R52E (labeled) and 30 μM 1-277_E17R (unlabeled). The protein mixture was incubated with DNA oligonucleotides [(GT)6: 70 μM, (GT)6 A12 (GT)6: 35 μM or (GT)12: 35 μM].

For NTD competition experiments, 30 μM 1-277_R52E (labeled) was mixed with 30 μM 1-277_E17R (unlabeled) and then incubated with DNA oligonucleotides [(GT)6: 70 μM, (GT)6 A12 (GT)6: 35 μM or (GT)12: 35 μM]. The complex mixture was incubated with 720 μM (24 times to 1-277_E17R) 1-93_E17R (unlabeled isolated NTD) in final.

All the samples were prepared in 12 mM phosphate at pH 6.9, containing 100 mM KCl and 2 mM TCEP. RNA-bound protein samples were supplemented with SUPERase·In RNase Inhibitors (Thermo Fisher Scientific). Samples were concentrated to a final volume of 180 μL, containing 10 μL D2O for locking procedure and then transferred into 3 mm diameter NMR tubes (NORELL) for experiment acquisition. 2,2-Dimethyl-2-silapentane-5-sulfonic acid was used as a reference in pure D2O (Eurisotop) for chemical shift referencing.

For NMR measurements, all the experiments were 2D SOFAST-HMQC experiments53. NMR spectra were acquired on a Bruker AVIII HD 600 MHz spectrometer equipped with a triple-resonance cryoprobe at 308 K and processed by Topspin 4.3.0 (Bruker). The number of scans and the relaxation delay corresponding to experiments involving in 1-277_WT or 1-93_WT were set to 1024 and 0.2 s. Experiments involving in mutants corresponded to a number of scans of 332 and a relaxation delay of 0.2 s. The number of scans and the relaxation delay of the experiments of 101-277_WT were 512 and 0.2 s. All the experimental data were acquired with 2048 and 128 complex pairs in direct 1H and indirect 15N. The corresponding bandwidth was 12.5 ppm (1H) and 34.5 ppm (15N). Shaped pulse length and power were calculated by considering an amide 1H bandwidth of 4.5 ppm and a chemical shift offset of 8.5 ppm.

For NMR analysis, CcpNmr AnalysisAssign 3.1.0 was used for NMR spectra assignment and analysis. The chemical shifts obtained from our NMR experiments are similar to those available in the BMRB database, which facilitated the assignments. The 1H and 15N chemical shift assignments of NTD-RRM1-2 fragments or RRM1-2 fragments were done by using published assignments for NTD and unbound RRM1-2 (BMRB Entries: 30345 and 27613, respectively). The 1H and 15N chemical shift assignments of NTD-RRM1-2 fragments or RRM1-2 fragments binding to (GT)-rich or (GU)-rich oligonucleotides were performed by using published assignments for NTD (BMRB Entry: 30345) and (AUG12)-bound RRM1–2 (BMRB Entry: 19290). All these BMRB entries are published in the Biological Magnetic Resonance Bank (BMRB) database and can be used for free. The chemical shift perturbations (CSPs) were calculated by the formula following Eq. (4):

Δδ= [0.5 * (ΔδH)2 + (0.14ΔδN)2] 0.5 (4)

where ΔδH and ΔδN are chemical shift variations in proton and nitrogen dimensions, respectively.

Small-angle X-ray Scattering (SAXS)
SAXS data were collected on SWING beamline at Synchrotron SOLEIL (Saint- Aubin, France)54. Data collection parameters are shown in Supplementary Table 2. All the measurements were performed in the SAXS measuring cell with a 1.5 mm diameter quartz capillary. The measurements of NTD-RRM1-2 fragments in presence of RNA or DNA oligonucleotides were performed by using an Agilent BioSec5-500 column. The concentration of proteins and oligonucleotides were reported in Supplementary Table 2. All the samples were prepared in 20 mM Hepes buffer at pH 7.6, containing 100 mM KCl and 2 mM TCEP. The samples in presence of RNA were supplemented with SUPERase·In RNase Inhibitors. In each experiment, 50 μL of sample was loaded into the column with a flow rate fixed at 0.3 mL/min at 25 °C. Scattering of the elution buffer before void volume was recorded and used as buffer scattering for subtraction from protein/oligonucleotide patterns.

2D SAXS images obtained were radial averaged and normalized by Foxtrot, a Swing in-house software, and the 1D curves were analyzed by BioXTAS RAW 2.2.155. An advanced Series processing in RAW–Regularized Alternating Least Squares (REGALS)56 was also used for some samples to decompose when peak overlapping occurred to eliminate the impact of higher-order oligomer contamination on the sample’s elution peak. Samples in the presence of (GT)19 or (GT)24 were separated by using REGALS. In this program, the radius of gyration (Rg) and I(0) were calculated via Guinier fit. The molecular weight was estimated based on the correlation volume (Vc)57. Maximal extension Dmax was calculated via GNOM58. All these structural parameters (radius of gyration Rg [Å], maximal extension Dmax [Å], and molar mass MM [kDa]) shown in Supplementary Table 2 were acquired from averaged SAXS experimental curves, which were generated by calculating an average of several SAXS curves with a similar and stable Rg values allowing a better signal/noise ratio. Dimensionless Kratky plots were used to better visualized the flexibility/extendedness of different protein/oligonucleotides complex59. The accuracy of the fit is evaluated using χ² values derived from the two equations below (5) and (6)60:

χ2= 1/N-1 Σ [Iexp(qj) - cItheor(qj) / σ(qj)]2 (5)

where N is the number of points in the experimental curve, Iexp(q) is obtained by experimental data, σ(q) are the experimental errors, Itheor(q) is the calculated theoretical intensity and c is the scaling factor given as in Svergun et al. (1995)61.

c = Σ [Iexp(qj) * Itheor(qj) / σ(qj)2] / Σ [Itheor(qj)2 / σ(qj)2] (6)

Construction of the 3D models of TDP-43 protein
3D models of wild type and R52E mutant TDP-43 protein (a.a., 1-277) have been generated with BIOVIA Discovery Studio Visualizer software (v24.1.0.23298, Dassault Systems, San Diego, 2023) using our model of TDP-43 RRM1-2 (a.a., 101-277) in complex with (GU) previously published24 and a TDP-43 NTD crystal structure (PDB 5MDI). The unfolded linker region (a.a., 80-100) has been added between these two structures.

DADIMODO calculations were performed using the webserver (https://dadimodo.synchrotron-soleil.fr). The two RRM domains (residues from 100 to 277) and the RNA were defined as a unique rigid body. The two NTD domain (residues 1 to 77), from each TDP-43 (a.a., 1-277) unit, were defined as separated rigid body.

Cross-linking Assay
100 pmol purified 1-277_WT or 1-277_R52E were incubated at 37 °C during 30 min in presence of 30 pmol of the oligonucleotide [GAAGAAGAGAAGAAGAAG-A6-(GT)12-A6-CTTCTTCTTCTCTTCTTC] named Stem-loop(GT)12 or 10 pmol of the oligonucleotide [GAAGAAGAGAAGAAG-(GT)12-A6-(GT)12-A6-(GT)12-CTTCTTCTTCTCTTCTTC] named Stem-loop(GT)12 A6 (GT)12 A6 (GT)12. The experiments were done in 20 mM Hepes buffer at pH 7.6, containing 25 mM KCl, 1 mM TCEP and 2 mM MgCl2. 1 mM BS3 chemical arm (bis(sulfosuccinimidyl)suberate) was added into the incubated samples for cross-linking reaction. The reaction last 20 min at room temperature and then 100 mM Tris-HCl (pH 8.0) was added into the mixture to stop the reaction and remove the excess of BS3. The mixture was incubated for 20 min at room temperature. The cross-linked proteins were separated by SDS-PAGE (10% poly-acrylamide) and visualized by Coomassie-blue.

Electrophoretic mobility shift assay (EMSA)
Previously purified TDP-43 fragments (a.a., 1-277) were incubated in the presence of 10 pmol of the oligonucleotides, stem-loop (GT)36, stem-loop A48 or stem-loop A72 (Sequences in Supplementary Table 3). The stem structure facilitates the ethidium bromide visualization on a poly-acrylamide gel. The mixtures were incubated with 20 mM Hepes buffer at pH 7.6, containing 25 mM KCl, 1 mM TCEP, and 2 mM MgCl2 at room temperature for 20 min in final volume of 20 μL. Free and bound to protein oligonucleotides were separated in an 8 % poly-acrylamide gel in 0.5× TAE buffer at 80 V for 85 min on ice. Finally, gels were stained with 0.5 μg/mL of ethidium bromide.

Cell culture and transfection
HeLa cells (obtained from ATCC, CCL-2) were cultured at 37 °C, 5% CO2 in high glucose DMEM medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Thermofisher), 100 units/mL of penicillin and 100 μg/mL of streptomycin (Life Technologies). Cells at confluence were plated in 96-well plates (PhenoPlate™, PerkinElmer) at a density of 18,000 cells per well and transfected with target plasmids (0.25 μg/well) or siRNA (0.2 μg/well) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Transfected cells were incubated for 24 h at 37 °C, 5% CO2, and processed (treated or fixed) depending on the assay.

Imaging parameters
There were no changes in the imaging parameters in the following assays. In addition, our pipelines from the cell detection to image analysis are entirely automated.

Microtubule bench assay
HeLa cells were co-transfected with the plasmids encoding RFP-MBD- or GFP-MBD-fused proteins as described above. The indicated plasmids were obtained previously25. Mutations were introduced by site-directed mutagenesis and were validated by DNA sequencing (Eurofins Genomics). Following 24 h cells were fixed with ice-cold methanol for 10 min at -20 °C, washed once with PBS, and further fixed with 4% paraformaldehyde (PFA) diluted in PBS for 10 min at 37 °C. The cells were washed three times with PBS and stained with 4 μg/mL DAPI to visualize the nuclei.

The cell images were obtained with the Opera Phenix™ Plus High Content Screening System (PerkinElmer) in confocal mode using a ×40 NA1.1 objective with water immersion.

Cell image analysis was performed using the Harmony v5.2 software (PerkinElmer) as described previously28. Briefly, we selected the population of co-transfected cells expressing both RFP- and GFP-fused proteins. Nuclei, cytoplasm, and microtubule clusters were detected automatically. Identified clusters of microtubules (spots) were then filtered by applying thresholds to remove non-tubular spots: spot area (>20 px2 and <300 px2) and spot width-to-length ratio (<0.3). The mean intensities of GFP and RFP in each selected microtubule cluster were measured and normalized to the mean fluorescence intensities in the cytoplasm. Mixing score (R-squared) was obtained by plotting the GFP vs RFP and applying a linear regression with automatic detection of R2 values. Data analysis, including filtering and calculations, was performed using MATLAB R2023a.

Nuclear aggregate assay
HeLa cells were transfected with the indicated plasmids encoding HA-fused proteins as described above. Plasmid harboring HA-TDP43 were obtained previously25. Mutations were introduced by site-directed mutagenesis and were validated by DNA sequencing (Eurofins Genomics). Following 24 h of transfection, cells were treated with the indicated concentration of arsenite or DMSO for 1 h at 37 °C. After treatment, cells were washed with PBS, and fixed with 4% PFA for 20 min at 37 °C. The cells were washed three times with PBS and incubated with the blocking solution (PBS, 2% BSA, 0.2% Triton X-100) for 1 h at room temperature. Next, cells were incubated with mouse monoclonal anti-HA antibodies F-7 (Santa Cruz Biotechnology, sc-7392, 1:1000) overnight at 4 °C with the following incubation with donkey anti-mouse DyLigh-488-conjugated secondary antibodies (Invitrogen, SA5-10166, 1:1000). In addition, rabbit polyclonal anti-G3BP1 antibodies (Sigma-Aldrich, G6046, 1:1000) and donkey anti-rabbit DyLigh-488-conjugated secondary antibodies (Invitrogen, SA5-10038, 1:1000) were used to detect G3BP1 and stress granules.

To visualize poly-A mRNA in HeLa cells, mRNA in situ hybridization was performed. Following the PFA fixation cells were incubated with ethanol 70% for 10 min at RT and with 1 M Tris-HCl pH 8.0 solution for 5 min. Next, cells were incubated with Cy3-conjugated oligo-dT probes (1 μg/mL, Molecular Probed Life Tech) in the hybridization buffer (0.005% BSA, 1 mg/mL yeast RNA, 10% dextran sulfate, 25% formamide in 2X SSC buffer). Finally, cell nuclei were stained using DAPI (4 μg/mL).

The cell images were obtained with the Operetta CLS™ High-Content Analysis System (PerkinElmer) in confocal mode using a ×20 NA1.0 objective with water immersion. Cell image analysis was performed using the Harmony v5.2 software (PerkinElmer). We selected the population of transfected cells expressing HA-fused proteins. HA-tag corresponds to a nona-peptide YPYDVPDYA derived from the hemagglutinin (HA) protein. This tag is used as antigen recognized by specific antibodies. Following nuclei detection using DAPI, granules of HA-tagged proteins in nuclear regions were detected automatically. The Harmony software calculated the relative intensity of nuclear aggregates as the ratio of (1) integrated signal from all pixels of all nuclear aggregates in a nucleus (intensity within the granule minus the local background intensity) and (2) integrated intensity over the nucleus. The enrichment of poly-A mRNA in nuclear aggregates was calculated as the ratio between the fluorescence of poly-A mRNA in a nuclear aggregate and the entire nucleus. Data analysis, including filtering and calculations, was performed using MATLAB R2023a.

Splicing reporter assay
HeLa cells were transfected with the TDP43-specific siRNA (5’-CACUAC AAU UGA UAU CAA A)TT-3’, Eurofins Genomics) and non-silencing siRNA (AllStars Negative Control QIAGEN, 10272281) as described above. Cells were incubated for 24 h and transfected with the indicated plasmids encoding splicing reporter minigenes. Following 24 h of the transfection cells were washed with PBS, and fixed with 4% PFA for 20 min at 37 °C. The cells were washed three times with PBS, incubated with the blocking solution for 1 h at room temperature, and then incubated with anti-HA antibodies (1:1000, Santa Cruz Biotechnology) overnight at 4 °C. Next, cells were incubated with goat anti-mouse IRDye® 680RD secondary antibodies (LI-COR, 926-68070, 1:1000) for 1 h, at room temperature, washed with PBS, and incubated with DAPI for nuclei detection. Silencing efficiency was verified using rabbit anti-TDP43 antibodies (Proteintech, 12892-1-AP, 1:1000) and goat anti-rabbit IRDye® 680RD secondary antibodies (LI-COR, 926-68071, 1:1000).

The cell images were obtained with the Operetta CLS™ High-Content Analysis System (PerkinElmer) in confocal mode using a ×20 NA0.4 objective. Cell image analysis was performed using the Harmony v5.2 software (PerkinElmer). Following nuclei detection using DAPI, fluorescent intensities of GFP and RFP in the nuclear regions were measured.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The role of RNA-binding proteins (RBP) in RNA metabolism is currently subject to numerous investigations with many\nquestions yet to be answered. Feng et al., carried out a detailed structural analysis of the RBP, TDP-43, using Nuclear\nMagnetic Resonance, Isothermal Titration Calorimetry, Small Angle X-Ray Scattering, and cellular experiments. They\nindicate the vital role nuclear interaction of intronic GU-rich RNA with TDP-43 N-terminal domain and RNA-recognition\nmotifs, plays in preventing TDP-43 aggregation, and important process in the pathology of neurodegeneration. Oxidative\nstress of cells upon exposure to arsenite induced TDP-43 aggregation modulated by GU-rich RNA. Finally, the authors work\nsuggested that stress conditions induced acetylation of TDP-43 in some RRM residues, leading to TDP-43 aggregation. The\nwork is interesting and builds off similar findings from other groups in this area over the last ~10 years, although there is\nsome over-interpretation of the cellular data which requires futher biochemical analysis to confirm findings.\nMajor Points\n• Introduction Line 60 – which part of TDP-43 the authors are referring to as the NTD is unclear. Stated ‘no RBP other than\nTDP-43 itself harbors a similar NTD domain’, this is true, although other RBPs have similar RRMs (albeit not within their\nNTDs, eg FUS has similar RRMs in its CTD). These descriptions should be more clear – it appears the authors refer to the\nNTD as the sequence N-terminal to RRM1 in later parts of the paper but this is vague in Introdution. NTD and RRM\nacronyms should be defined in abstract.\n• Fig 1A is not to scale with a comparatively shortened CTD shown; this should be clarified.\n• TDP-43 clearly preferentially binds to GU-rich RNA. The authors could consider showing control data (separate from the\npolyA sequence already provided) indicating how CA-rich RNA (usually used as control for GU RNA in previous studies)\ninfluences TDP-43 RRM1=RRM2 interaction and interactions between the NTDs of TDP-43 monomers.\n• The authors use either the E17R or R52E mutant to prevent intermolecular association between TDP-43 NTD. However\nprevious reports have used 6-point mutations in the NTD to prevent TDP-43 oligomerisation – 6M (E14, E17, E21, Q34, R52\nand R55). The authors should show data that the single R52 mutant is sufficient to completely prevent TDP-43 interaction\nand how this compares to 6M mutant to allow for comparison to previous studies. Otherwise, the authors should include\nsome 6M data in their RNA NMR experiments, or discuss differences with the previous literature.\n• The SAXS data observation of compacted nucleoprotein complex is only observed in the truncated TDP-43 NTD/RRM\n(Fig. 3). The authors should conjecture on how this is translated to full length TDP-43 compaction in these RBP complexes.\n• Data presentation is sometimes confusing an – eg Fig 4C,D,E y axes start at non-zero which over-conflates the changes\nseen, and arrows drawn on graphs are not needed. %change on graph is not informative, instead should report on statistical\nanalyses. Also, the 8 datapoints appear to be technical rather than experimental replicates (stated that each is a separate\nwell) – these data should be analysed with biological replicates from separate experiments conducted independently.\n• Fig 5 shows effects of arsenite on TDP-43 aggregation, which is usually taken as a biochemically determined feature – to\nconfirm ‘aggregation’, authors should perform experiemnts to determine whether there are differential effects on\nsoluble/insoluble protein ratio of mutants with arsenite treatment. This is particularly important given the focus on ‘soluble\nproteins’ (the data presented does not in fact investigate solubility), and also as there appears to be some difference in\nimage quality (eg HA-FUS in Fig 5B is clearly not the same resolution/focus as HA-TDP-43) which could account for the\nlack of detection of puncta in response to stress. Were all images collected using the same parameters? This issues is also\nsomewhat confounded in the text with interchangeable use of the term ‘granule’ in the figures/methods and ‘aggregate’ in\nthe text (eg Supp Fig 14 – are the authors refering to 2 different structures or are they the same, and what are the definitions\nof granule vs aggregate?) – closer attention to consistency would assist the reader interpret findings. Without biochemical\nconfirmation, the focus on solubility and aggregation is weak.\n• The authors could further explore how GU-rich RNA TDP-43 interactions intersects with TDP-43 acetylation that has been\ndetected in TDp-43 pathology related to aggregation to provide even further disease context by using the K145Q and K192Q\nacetylation-mimic mutants that recapitulate changes seen in ALS spinal cord pathology (in addition to the data on K-R\nacetylation blocking mutants).", "reviewer_2": "The manuscript by Feng et al. provides important mechanistic insights into how TDP-43, a key RNA-binding protein\nimplicated in ALS and FTLD, forms both physiological and pathological assemblies. Extending their previous work, the\nauthors systematically examine the interplay between TDP-43’s N-terminal domain (NTD)-mediated self-association and its\ncooperative binding to GU-rich RNA, employing a combination of biochemical, structural (NMR, SAXS), and thermodynamic\n(ITC) approaches, along with cellular assays.\nTheir premise is that under normal nuclear conditions, TDP-43 binds cooperatively along GU-rich intronic RNA, suppressing\naberrant NTD:NTD interactions while promoting RNA compaction through long-range clustering of TDP-43:RNA complexes.\nIn contrast, under oxidative stress or in the cytoplasm, where GU-rich RNAs are limited, disruption of cooperative RNA\nbinding permits pathological NTD:NTD interactions that drive TDP-43 aggregation. Moreover, they show that expression of\nGU-rich RNA can protect against stress-induced aggregation.\nThis work highlights the dual role of TDP-43’s NTD and RNA-binding domains in both functional RNA processing and\npathological aggregation. The authors present an impressively large amount of high-quality, carefully controlled data, which\nthey meticulously analyze to support their model. However, several questions regarding their interpretation need to be\naddressed before the manuscript is suitable for publication.\nMajor Points:\n1. The NMR line-broadening data supporting the hypothesis that cooperative binding of the RRMs to GT12 DNA prevents\nNTD interactions are not as convincing as claimed. The authors focus primarily on residues that support their model (e.g.,\nFigs. 2, S5, S6, S8). What about other key interface residues such as R52, E17 (a particularly critical residue), R55, G53,\nand T30? For example, in Fig. S8, while T32 and E21 show increased intensity in GT12-bound samples, I18 and I16 do not.\nAdditionally, the molecular size differences between GT6- and GT12-bound samples complicate direct intensity\ncomparisons (e.g., E156, which likely does not engage in NTD interactions, varies between conditions). Some affected\npeaks remain unlabeled, such as a Trp indole NH and a peak near 6.3 ppm.\n2. Even if the authors can convincingly demonstrate that peak intensities are partially recovered in the presence of GT12,\nthe interface residue signals do not fully return to levels observed in the absence of NTD interactions, suggesting that\nsignificant NTD interactions persist when bound to GT12 (Fig. 2B). The manuscript overemphasizes these findings, and together\nwith the SAXS data, seems to suggest that cooperative RNA binding completely eliminates NTD:NTD interactions, which is not\nfully supported by the data.\n3. It remains unclear why the NTD would be unable to engage in intermolecular interactions when TDP-43 is complexed\nwith DNA/RNA (GT6 or GT12). The authors provide evidence that the NTD does not directly bind RNA or the RRMs and that\nthe RNA:TDP-43 complexes are monodisperse by SEC. What, then, prevents NTD self-association under these conditions?\nAlternatively, are NTD:NTD interactions simply transient, only becoming significant at higher local concentrations, such as\nwhen multiple TDP-43 protomers are bound to RNA?\n4. In the SAXS model presented in Fig. S11, the two NTDs appear close enough to interact. Additional analysis such as\nconsidering linker lengths, distances between RRM1/2 domains, and RRM dimensions would strengthen the argument that\nNTDs remain separated. Furthermore, the SAXS data suggest the presence of multiple conformations, making it potentially\ninappropriate to present a single “best-fit” model as definitive.\n5. Analyzing the populations of bound and unbound states would better demonstrate whether, in the cooperatively bound\nsamples, NTDs are largely unassociated. Such analysis could also help resolve concerns mentioned in the points above.\nAdditionally, the authors should discuss (or quantify) the dynamic nature of NTD interactions in detail, as their own NMR\ndata (e.g., peak splitting in Figs. 2, 6, and 8) suggest the presence of multiple conformations.\n6. Statements suggesting that \"cooperative binding of TDP-43 to long (GT12) repeats weakens interactions between NTDs\"\n(e.g., p.6, line 254) are potentially misleading. These phrases imply direct regulation of NTD association by RNA binding,\nyet the data do not clearly support a direct mechanistic link. Such statements should be revised for clarity and accuracy.\n7. The model shown in Fig. 7B is misleading, as it does not clearly depict why NTDs from adjacent TDP-43 protomers\ncannot interact. The figure should be revised for clarity.\nMinor Points:\n8. Line 374 – \"act together\" should be replaced with \"each contribute.\"\n9. Some sections of the manuscript are repetitive and could be streamlined to improve readability.\n10. The physical meaning of negative chemical shift perturbations (CSPs) in Fig. 1 and S1 is unclear. Correlation plots\ncomparing CSPs between conditions may be more effective in illustrating differences and avoiding confusion.\n11. The main text figures should include error estimates for ITC-derived KD values.\n12. The equations used for fitting the ITC and SAXS data should be included in the Methods section.\n13. Figures are referenced out of order in some sections (e.g., Fig. S17 etc.).\n14. Some reported fitting errors for the ITC data (Table S2) seem small and should be double-checked.\n15. The manuscript would benefit from thorough editing to correct pluralization errors and missing articles throughout the\ntext.", "reviewer_3": "The binding studies are particularly interesting and provide new mechanistic insights into TDP-43 assembly. The results are\nclearly presented, supported by multiple complementary methods, and I highly recommend this manuscript for publication.\nSome minor issues\n1) SAXS-related issues\n1.1 Please deposit (at least WT SAXS data) in the SASBDB.\n1.2 in General consider showing p(r) functions when discussing the changes in size. Kratky plots are shown throughout the\nmanuscript but the information gained from the plot is not stated.\nEg. in line 277: Reference to SupplementFigure 9B is misleading at this point -- this is Kratky Plot and does not refer to\nDmax. Here comparison of P(r) funtiontions would emphasize this point (misleading figure legend in Sup FS9B,\nE17R+R52E, but not E17R alone, reduced the sizes of the complexes in the presence of (GT)6A12(GT)6.\n1.3 In general, the SAXS results could be strengthened by including quantitative comparisons—for example, reporting the\nDmax: for wild-type TDP-43 (162 nm) alongside the range observed for the mutants (159–165 nm). --> and quantify\naccordingly in line 286 as well the increase in Dmax values (line 297) and later in text. Is there a way to estimate the errors\nfrom Dmax? (Obviously the information is given in various tables, but could help the reader).\n1.4 Elution profiles\nSupplementary Figure S9C -- Please comment on the shoulders - such as for the E17R mutants\nSupplementary Figure 11 -- Comment on the shoulders at frame 100 -- are these aggregates - higher oligomeric structurs?\nMinor (SAXS section):\n• line 265\n• The chromatograms and the SAXS images were recorded during the elution. We observed that the elution volumes were\nsimilar for all the conditions in the presence of (GT)12 for which the binding is cooperative.\"\n• To improve clarity, consider explaining the underlying observations in a bit more detail\neg. The single mutants (blue and yellow trace) elute similarly as WT (red trace), which shows cooperative binding (see ITC\nFig 1D, red plot)\n• Supplementary Figure S9A -- I'm not sure what the difference is between the upper and lower panels. Perhaps a\ncopy/paste issue with the legend?\n• line 260 -- Restructure: To investigate structural changes on a larger scale than accessible by NMR, we performed SAXS\nanalysis of in vitro–purified TDP-43–nucleic acid complexes.\n• line 264: The acquisition of the SAXS images WAS..... or SAXS measurements were performed in-line with size-exclusion\nchromatography (SEC)\n• line 265 -- specify which chromatgram (UV 280nm + 260nm ?)\n• line 273 : Already here, place the reference to Figure 3 and SAXS Table in supplementary information. (see line 277)\n• line 274: As two hypotheses were formulated earlier be specific which one is in agreement.\n• Line 1940 -- in Methods the use of REGALS for decomposing overlapping peaks is mentioned -- was this observed in the\nmeasurements?\n• Line 1945 -- not clear what is meant with: were acquired from averaged SAXS experimental curves.\n• Line 1950 -- models ... HAVE been generated\nGeneral:\n• To improve clarity and conciseness, consider introducing the E52R and R17E mutants, along with the different nucleotide\nconstructs\nsuch as (GT)₆A₁₂(GT)₆, earlier in the text. This would reduce the need for repeated reintroductions later in the manuscript.\n• line 277m, replace \"Accordingly\" with 'next', 'in addition', ...\n• line 19 -- not clear which mechanisms are being referred to\n• line 29 -- define RRM at this point\n• line 30 -- is it assembly of TDP-43 in (OR ON ?) introns\n• line 144 -- better: .. and includes the full NTD domain. (instead of while keeping)\n• line 148 -- ..we compared the chemical shifts of the RRM1-2 residues to contstucts containing (1-277) and lacking (101-\n277) the NTD domain\n• line 167 -- replace whatever with whether\n• line 170 -- In THE presence of...\n• line 172 -- remove 'a' to read: a major feature of cooperative association with (GT)₁₂\n• line 177 -- perhaps add 'red plot (Figure 1D)\n• line 178 -- why not mention the second mutant?\n• line 183 -- add \"green plot\"\n• line 214 -- 'A' similar result...\n• line 267 -- add: the binding is cooperative (WT AND SINGLE MUTANTS).\n• line 290 -- remove 'The' before 'similar results'\n• line 317 -- replace 'To document' with to assess or investigate or characterise...\n• line 553 -- mutated TDP/43*aa. 1/277\n• line 382 -- define 'HA'\n• line 401 -- use passive form : Two pairs of lysine residues, known to undergo acetylation following arsenite stress, were\nmutated to arginine.\n• Supplementary Figure 2B: One could first show R52E mutant then the double mutant\n• Supplementary Figure 3: Color names should be in lowercase rather than uppercase.\n• line 1089 -- interactions DO not interfere\n• line 1098 -- Significant CSPs ARE OBSERVED only in the binding residues of RRM1-2 (assigned in black).\n• line 1099 -- COMPARED to ....\n• line 1142 -- Zoomed-in VIEW on THE chemical shift perturbations FOR TDP-43 residues in the RRM, with or without the\nindicated nucleotidess\n• line 1144 -- R52E not R525E\n• line 1927 -- Data collection parameters ARE shown\n• line 1934 -- In each experiment -- (remove 's')", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Q1: Introduction Line 60 – which part of TDP-43 the authors are referring to as the NTD is\nunclear. Stated ‘no RBP other than TDP-43 itself harbors a similar NTD domain’, this is true,\nalthough other RBPs have similar RRMs (albeit not within their NTDs, e.g. FUS has similar\nRRMs in its CTD). These descriptions should be more clear – it appears the authors refer to the\nNTD as the sequence N-terminal to RRM1 in later parts of the paper but this is vague in\nIntroduction. NTD and RRM acronyms should be defined in abstract.\nWe agree with the reviewer. We indicated the meaning of the acronyms in the abstract. In\naddition, we added a sentence in the text to clarify the position of the NTD domain (Page 2, lines\n59-61).\nQ2: Fig 1A is not to scale with a comparatively shortened CTD shown; this should be clarified.\nThank you for noticing this. Figure 1A has been corrected.\nQ3: TDP-43 clearly preferentially binds to GU-rich RNA. The authors could consider showing\ncontrol data (separate from the polyA sequence already provided) indicating how CA-rich RNA\n(usually used as control for GU RNA in previous studies) influences TDP-43 RRM1=RRM2\ninteraction and interactions between the NTDs of TDP-43 monomers.\nWe agree with the reviewer. In the revised manuscript, we provided new data to answer this\npoint. In the Supplementary Figure 5, new EMSA results show that wild-type or mutant forms do\nnot bind to A nor A compared to (GT) oligonucleotide. Moreover, NMR results show no\n48 72 36\nsignificant shift observed in spectra of wild type or mutant in presence A or (CA)\n12 6\noligonucleotides, comparing to protein alone. Therefore, TDP-43 poorly interacts with poly A\nand CA-rich oligonucleotides compared to GT repeats.\nA comment has been added to text to present these additional controls (Page 5, lines 183-186).\nQ4: The authors use either the E17R or R52E mutant to prevent intermolecular association\nbetween TDP-43 NTD. However, previous reports have used 6-point mutations in the NTD to\nprevent TDP-43 oligomerisation – 6M (E14, E17, E21, Q34, R52 and R55). The authors should\nshow data that the single R52 mutant is sufficient to completely prevent TDP-43 interaction\nand how this compares to 6M mutant to allow for comparison to previous studies. Otherwise, the\nauthors should include some 6M data in their RNA NMR experiments, or discuss differences\nwith the previous literature.\nWe understand the point raised by the reviewer. However, in our hand, a single mutation, R52E\nor E17R, in the NTD was enough to significantly disrupt NTD/NTD interactions. Indeed, the\nelution of 1-277_WT, 1-277_E17R and 1-277_R52E by a Size Exclusion Chromatography\n(SEC) column was shown in the new Supplementary Figure 1. Mutant E17R and mutant R52E\ndisplay lower elution volumes than wild type TDP-43 due to the disruption of NTD/NTD\ninteractions. The molecular weight (MW) of R52E mutant, E17R mutant and WT predicted by\nSAXS experiments are 34.5kDa, 34kDa and 44.1kDa, respectively. The predicted MW of R52E\nand E17R is much closer to the theoretical value (MW 31.5kDa) than WT. Moreover, we\nobserved larger aggregates during the elution of wild type TDP-43 than two single mutants.\nQ5: The SAXS data observation of compacted nucleoprotein complex is only observed in the\ntruncated TDP-43 NTD/RRM (Fig. 3). The authors should conjecture on how this is translated to\nfull length TDP-43 compaction in these RBP complexes.\nIndeed, we used truncated TDP-43 in vitro because the unstructured CTD leads to TDP-43\naggregation in vitro. TDP-43 CTD may also contributes to the compaction of nucleoprotein\ncomplex through addition homotypic interactions. In addition, TDP-43 CTD may also enable\nheterotypic interactions with protein partners in the nucleus. A comment has been added in the\ndiscussion (Page 12, lines 508-513).\nQ6: Data presentation is sometimes confusing an – eg Fig 4C,D,E y axes start at non-zero which\nover-conflates the changes seen, and arrows drawn on graphs are not needed. %change on graph\nis not informative, instead should report on statistical analyses. Also, the 8 datapoints appear to\nbe technical rather than experimental replicates (stated that each is a separate well) – these data\nshould be analyzed with biological replicates from separate experiments conducted\nindependently.\nWe agree that the scales shown in Figure 4 do not start at zero. We did this to facilitate the\nreading and visualize the variations. This is why we would like to ask the reviewer if we could\nkeep this representation of the data. The values shown correspond to the R² value. For all the\nvariations mentioned in the text, we verified their significance through statistical analysis.\nRegarding the experimental replicates, we now provide at least one replicate obtained on\nindependent plates and performed on a different week. The analyses for experimental replicates\nhave been added in the Supplementary Figure 20.\nQ7: Fig 5 shows effects of arsenite on TDP-43 aggregation, which is usually taken as a\nbiochemically determined feature – to confirm ‘aggregation’, authors should perform\nexperiemnts to determine whether there are differential effects on soluble/insoluble protein ratio\nof mutants with arsenite treatment. This is particularly important given the focus on ‘soluble\nproteins’ (the data presented does not in fact investigate solubility), and also as there appears to\nbe some difference in image quality (eg HA-FUS in Fig 5B is clearly not the same\nresolution/focus as HA-TDP-43) which could account for the lack of detection of puncta in\nresponse to stress. Were all images collected using the same parameters? This issues is also\nsomewhat confounded in the text with interchangeable use of the term ‘granule’ in the\nfigures/methods and ‘aggregate’ in the text (eg Supp Fig 14 – are the authors refering to 2\ndifferent structures or are they the same, and what are the definitions of granule vs aggregate?) –\ncloser attention to consistency would assist the reader interpret findings. Without biochemical\nconfirmation, the focus on solubility and aggregation is weak.\nTDP-43 aggregation after RRM acetylation upon arsenite exposure in cells has been already\nreported in the literature (Ref 26, Cohen et al, Nature Com, 2015).\nWe agree about the point raised by the reviewer to provide additional controls. We performed\ntwo experiments to explore the reversibility of TDP-43 assemblies in the nucleus after arsenite\ntreatments. First, we considered whether nuclear TDP-43 could be extracted thought detergent\ntreatment to briefly permeabilize the cell membrane after arsenite treatments. In the new\nSupplementary Figure 22, after washing with 0.1% Triton x100 for 5 min, we found nuclear\nTDP-43 levels were little affected for WT and G146A, but decreased for mutants R52E-G146A\nand R52E. This is in agreement with a higher solubility of R52E-G146A and mutant R52E than\nWT and G146A upon arenite treatment.\nSecond, in the new Supplementary Figure 23, we tested the reversibility of nuclear TDP-43\naggregates/granules formed after arsenite treatments. To this end, after arsenite treatments,\narsenite was washed out for 90 min. As expected, we found that G3BP1 stress granules (here 200\nµM of arsenite was used to trigger a robust stress granule assembly) dissociate completely in\nagreement with the reversible nature of these RBP-rich condensates. In contrast,\naggregates/granules of TDP-43_WT and TDP-43_G146A formed with 80 µM of arsenite are still\nthere, even though we evidenced a partial dissociation (which may be due to the partial removal\nof aggregates by cells during 90 min). Therefore, we considered TDP-43 aggregates/granules\nafter arsenite treatment as aggregates in the text (though they may as well be solid-like\ncondensates of very low kinetics). This result is in line with the previous knowledge of TDP-43\naggregation upon arsenite treatments and with the absence of mRNA enrichment in these\nassemblies (Figure 5A).\nRegarding the suggestion to perform biochemical analyses, we think this hardly possible with\nTDP-43 since it has a strong tendency for aggregation in vitro after cell lysis. Therefore, we\nthink that cell assays are more informative concerning this point than biochemical assays.\nIn response to Q7, two new Supplementary Figures 22 and 23 have been added to manuscripts\nand their results commented in the text and supplementary figure legends (Page 10, lines 419-\n427).\nQ8: The authors could further explore how GU-rich RNA TDP-43 interactions intersects with\nTDP-43 acetylation that has been detected in TDP-43 pathology related to aggregation to provide\neven further disease context by using the K145Q and K192Q acetylation-mimic mutants that\nrecapitulate changes seen in ALS spinal cord pathology (in addition to the data on K-R\nacetylation blocking mutants).\nThis is an interesting point. We generated the K145Q and K192Q acetylation-mimic mutants.\nThe results have been added in the Supplementary Figure 24A. After arsenite treatment,\nMutants K145Q-K192Q or G146A-K145Q-K192Q form nuclear aggregates. In contrast, nuclear\naggregation of TDP-43 in cells expressing R52E-K145Q-K192Q or R52E-G146A-K145Q-\nK192Q is limited. Therefore, the acetylation of RRM lysine residues contributes to TDP-43\naggregation. A comment has been added in the text (Page 10, lines 429-436).\nReviewer 2:\nQ1: The NMR line-broadening data supporting the hypothesis that cooperative binding of the\nRRMs to GT12 DNA prevents NTD interactions are not as convincing as claimed. The authors\nfocus primarily on residues that support their model (e.g., Figs. 2, S5, S6, S8). What about other\nkey interface residues such as R52, E17 (a particularly critical residue), R55, G53, and T30? For\nexample, in Fig. S8, while T32 and E21 show increased intensity in GT12-bound samples, I18\nand I16 do not. Additionally, the molecular size differences between GT6- and GT12-bound\nsamples complicate direct intensity comparisons (e.g., E156, which likely does not engage in\nNTD interactions, varies between conditions). Some affected peaks remain unlabeled, such as a\nTrp indole NH and a peak near 6.3 ppm.\nWe understand the point raised by the reviewer. In Figure 2A, we analyzed and measured the\nvariations in CSPs when R52E and wild-type TDP-43 interacts with (GT) or (GT) . For these\n6 12\nexperiments, we agree that the analyzes of the peak heights are more complicated to analyze than\nCSPs because of the difference of molecular weight in the presence of (GT) versus (GT) ,\n12 6\nwhich may explain why some residues such as I18 and I16 do not show increased intensity in the\npresence of (GT) .\nincluding E17, R55, G53, that were missing as suggested by the reviewer (Supplementary Figure\n11 and Figure 2A). These residues show more significant chemical shifts when WT (a.a. 1-277)\nbinds to (GT) than when WT binds to (GT) . In contrast, the same residues do not show any\n12 6\nchemical shifts in any condition for the mutant R52E. This is in line with our hypothesis. A\nsentence has also been added to the text (Page 6, lines 226-229). R52E was not shown because\nthe analysis is based on comparing the CSPs between wild-type TDP-43 and R52E for which\nR52 is mutated into E. T30 CSPs cannot be observed because of the superimposed presence of\nadditional peaks such as that of R171.\nIn addition, let us note that we performed competition assays with E17R (Figure 2D). Here we\nshow the relative variations in peak height for all the NTD residues of TDP-43 R52E mutations.\nThe normalization of the variations of the peak heights allows to withdraw the bias due to the\nmolecular weight differences when TDP-43 interact with (GT) A (GT) or (GT) (Figure 2D).\n6 12 6 12\nThe analysis of the ratio of peak heights in Figure 2D, indicate that I18, I16, I21 but also S29,\nV31 and T32 showed an increase in peak height in (GT) -bound samples compared to\n12\n(GT) A (GT) when E17R NTD was added. These residues are located just in front of R52 in\n6 12 6\nthe NTD dimer (Figure 2C), so that these results make sense. Residues that do not participate in\nthis interface do not show a relative increase in peak heights. It is also the case for the residues\nthat are located in the opposite interface around R52E. Let us remind that this R52E-related\ninterface is no longer functional because the mutated interface (E52-related interface) in the\nR52E mutant (15N-labelled TDP-43, a.a. 1-277) cannot interact with the mutated interface (R17-\nrelated interface) in E17R NTD (unlabeled NTD). Indeed, these respective substitutions\n(changing both the charge and the size of initial residue) hinders the constitution of the interface.\nOnly the E17-related interface in the R52E mutant can interact with the R52-related interface in\nE17R NTD.\nRegarding W68, it is not directly involved in NTD/NTD interactions (Wang et al, EMBO, 2018).\nAccordingly, we did not observe any relative increase in peak height ratio for the W68 backbone\n(Figure 2D). However, indeed, as noticed by the reviewer, there is an unexpected increase in\nCSP of this residue in wild type TDP-43 in the presence of (GT) in comparison with (GT)\n12 6\n(now indicated in Supplementary Figure 11). Since W68 is located in a flexible domain, the side\nchain may be sensitive to change in local structural environment. Regarding E156, E156 is\nlocated in a different domain (RRM1) far from the NTD interface in RRM1, which may be as\nwell sensitive to change in local environment. We now indicated that we focus our attention on\nNTD residues for the results presented in Figure 2 (Page 5, lines 213-215). The reviewer also\nmentioned a peak near 6.3 ppm. In Fig. S8 (now Supplementary Figure 12), in the presence of\noligonucleotide, the peak near 6.3 ppm corresponds to F229, which is not located in the NTD but\nin RRM2.\nQ2: Even if the authors can convincingly demonstrate that peak intensities are partially\nrecovered in the presence of GT12, the interface residue signals do not fully return to levels\nobserved in the absence of NTD interactions, suggesting that significant NTD interactions persist\nwhen bound to GT12 (Fig. 2B). The manuscript overemphasizes these findings, and together\nwith the SAXS data, seems to suggest that cooperative RNA binding completely eliminates\nNTD:NTD interactions, which is not fully supported by the data.\nWe agree with the reviewer that the peaks do not recover their initial height, which seems to\nindicate that interactions between NTDs still take place. We tuned down our conclusion and\nindicated that TDP-43 cooperative assembly in the presence of (GT) /(GU) reduces\n12 12\nNTD/NTD interactions instead of totally suppressing them.\nHowever, the signals of NTD residues detected by NMR represent an average value of several\ndynamic interactions over time. For example, in the presence of (GT) , one TDP-43 protein can\n12\ntransiently be released from RNA/DNA to initiate NTD/NTD interactions with an adjacent TDP-\n43. Therefore, even if the peak intensities of NTD residues do not fully return to their initial\nlevels corresponding to the absence of NTD/NTD interactions, we cannot exclude nor affirm that\nTDP-43 cooperative binding totally prevent NTD interactions after the analysis of this\nexperiment. We can just indicate that cooperative TDP-43 association to RNA/DNA limits\nNTD/NTD interactions.\nQ3: It remains unclear why the NTD would be unable to engage in intermolecular interactions\nwhen TDP-43 is complexed with DNA/RNA (GT6 or GT12). The authors provide evidence that\nthe NTD does not directly bind RNA or the RRMs and that the RNA:TDP-43 complexes are\nmonodisperse by SEC. What, then, prevents NTD self-association under these conditions?\nAlternatively, are NTD:NTD interactions simply transient, only becoming significant at higher\nlocal concentrations, such as when multiple TDP-43 protomers are bound to RNA?\nAccording to our model, the separation distance between the NTDs of adjacent TDP-43 units is\nlarge (Supplementary Figure 16, see text in Page 7, lines 285-288). Their interaction is therefore\npossible, as indicated by the reviewer, but it would occur at the expense of an entropy penalty.\nWe agree with the reviewer. NTD/NTD are indeed transient and the occurrence of NTD/NTD\ninteractions increases at high concentration (Ref. 18, Wang et al, EMBO, 2018). If a single TDP-\n43 monomer is bound to (GT) , the local concentration of NTD does not increase in the test tube.\n12 12\nwe may expect that the chances of having NTD/NTD interactions should increase. However, the\npoint is that we observed that the cooperative association of TD-43 to (GT) /(GU) negatively\n12 12\naffects NTD/NTD interactions (Figure 2D) and vice versa (Figure 1D).\nHowever, in agreement with the point of the reviewer, multiple TDP-43 bound to mRNA may\nprovide an additional contribution to explain why the cooperativity-deficient mutant, G146A, is\nparticularly prone to aggregation. Indeed, the binding of multiple G146A to long mRNA is\npossible which should promote the chances of NTD/NTD interactions between nearby TDP-43\nin the absence of cooperativity (free from the structural constraints imposed by the\ncooperativity).\nQ4: In the SAXS model presented in Fig. S11, the two NTDs appear close enough to interact.\nAdditional analysis such as considering linker lengths, distances between RRM1/2 domains, and\nRRM dimensions would strengthen the argument that NTDs remain separated. Furthermore, the\nSAXS data suggest the presence of multiple conformations, making it potentially inappropriate\nto present a single “best-fit” model as definitive.\nWe agree with the reviewer. They could interact but the unstructured linker between NTD and\nRRM1 will be stretched, which may lead to an entropy penalty. All the calculations performed\nusing DADIMODO resulted in good fits to the SAXS data, with χ2 values around 1.3–1.4. In all\nthe unique solutions obtained, the NTD domains were positioned far apart, with distances\nranging from 80 to 100 Å. The length of the linker between the RRM and NTD domains was\nfixed at 12 residues (residues 94 to 105). The superimposition of the five best results has been\nadded to the Supplementary Figure 16.\nWe agree that more than a single “best model” should be showed. The DADIMODO software\nindeed provides a single representative model that best fits the SAXS curve. These results\nindicate that a unique model with the two NTDs interacting directly is not compatible with the\nSAXS data. However, SAXS cannot fully exclude the presence of a minor population of\nconformations in solution where the NTDs are close. This point is detailed below in the response\nto question 5.\nA comment has been added regarding this point (Page 7, lines 289-294).\nQ5: Analyzing the populations of bound and unbound states would better demonstrate whether,\nin the cooperatively bound samples, NTDs are largely unassociated. Such analysis could also\nhelp resolve concerns mentioned in the points above. Additionally, the authors should discuss (or\nquantify) the dynamic nature of NTD interactions in detail, as their own NMR data (e.g., peak\nsplitting in Figs. 2, 6, and 8) suggest the presence of multiple conformations.\nWe agree with the reviewer and generated two closed state models displaying NTD/NTD\ninterface between adjacent TDP-43 along (GU) , with distance constraints (4-5 Å) between R52\n12\nresidue from NTD of one TDP-43 unit, and E17 residue from NTD of the second TDP-43 unit,\nand compared them to the experimental curve (Supplementary Figure 17).\nThe best-fitting models are linked to significantly elevated χ2 values, 3.12 or 3.60, when R52\n(from the first unit) interacts with E17 (from the second unit) or vice versa (E17/R52),\nrespectively, which are obviously worse than the value from the open state model (χ2= 1.35).\nThen, the best-constrained model (χ2= 3.12) was subsequently used as the starting point for a\nnew calculation performed without any distance constraints by using DADIMODO program.\nAccording to the new calculations, the best conformation was obtained, with a χ2 value of 1.33\nsimilar to the initial calculations (Supplementary Figure 16), corresponds to an open model in\nwhich the two NTD domains have moved apart (have separated) by approximately 72Å. We\nwould like to note that all of the best-generated solutions displayed the models where the two\nadjacent NTDs are separated. This analysis has been added in text (Page 7, lines 289-302). We\nthank the reviewer for his comment.\nIn summary, even if NMR and SAXS experiments are performed under different conditions, the\nanalysis of these data indicate that the cooperative association of TDP-43 to RNA limits\nNTD/NTD interactions.\nQ6: Statements suggesting that \"cooperative binding of TDP-43 to long (GT12) repeats weakens\ninteractions between NTDs\" (e.g., p.6, line 254) are potentially misleading. These phrases imply\ndirect regulation of NTD association by RNA binding, yet the data do not clearly support a direct\nmechanistic link. Such statements should be revised for clarity and accuracy.\nWe agree with the reviewer. The sentence has been rephrased: “NTD/NTD interactions between\nadjacent TDP-43 are limited when TDP-43 binds cooperatively to long (GT) repeats.” (Page 6,\n12\nlines 255-256). Many statements have been changed in the text to clarify this point.\nQ7: The model shown in Fig. 7B is misleading, as it does not clearly depict why NTDs from\nadjacent TDP-43 protomers cannot interact. The figure should be revised for clarity.\nWe agree with reviewer. Figure 7b has been revised. We indicate the large separation distance\nbetween NTD of adjacent TDP-43 limits NTD/NTD interactions.\nQ8: Line 374 – \"act together\" should be replaced with \"each contribute.\"\nWe agree with reviewer. It has been changed (Page 9, line 393).\nQ9: Some sections of the manuscript are repetitive and could be streamlined to improve\nreadability.\nWe thank to the reviewer. We tried to improve the clarity of the manuscript to the best we can.\nQ10: The physical meaning of negative chemical shift perturbations (CSPs) in Fig. 1 and S1 is\nunclear. Correlation plots comparing CSPs between conditions may be more effective in\nillustrating differences and avoiding confusion.\nWe agree with the reviewer. In the revised manuscript, we compared CSPs related to RRM1-2\nbetween R52E and E17R+R52E (Figure 1B), R52E and WT (Supplementary Figure 2B). No\nsignificant changes for most of the residues in RRM1-2, so NTD/NTD interactions do not affect\nRRM domains.\nQ11: The main text figures should include error estimates for ITC-derived KD values.\nWe agree with the reviewer. Error estimates for ITC-derived K values are now shown in Figure\nD\n1C and D.\nQ12: The equations used for fitting the ITC and SAXS data should be included in the Methods\nsection.\nWe agree with the reviewer. In ITC experiments, samples in the presence of (GT) and\n6\n(GT) A (GT) were fitting with the one set of sites model, while samples in the presence of\n6 12 6\n(GT) were fitting with the two set of sites model. The equations underlying these two fitting\n12\nmodels are described in detail in MicroCal PEAQ-ITC Analysis Software user manual. A\ndescription and the link of the user manual has been added in the text (Page 69, lines 2444-\n2454). The equations used for fitting the SAXS data have been added in the text (Page 71, lines\n2529-2536).\nQ13: Figures are referenced out of order in some sections (e.g., Fig. S17 etc.).\nWe agree with the reviewer. We changed the order of Supplementary Figures.\nQ14: Some reported fitting errors for the ITC data (Table S2) seem small and should be double-\nchecked.\nWe have double-checked the values. These values are estimated from the analysis by using\nMicrocal PEAQ-ITC Analysis Software provided by the manufacturer.\nQ15: The manuscript would benefit from thorough editing to correct pluralization errors and\nmissing articles throughout the text.\nWe are sorry for that. We tried to do our best to correct these errors.\nReviewer 3:\nQ1: Please deposit (at least WT SAXS data) in the SASBDB.\nWe thank to the reviewer. The SAXS data has been deposited on the SAXBDB database with the\naccession code SASDXL8.\nQ2: In General consider showing p(r) functions when discussing the changes in size. Kratky\nplots are shown throughout the manuscript but the information gained from the plot is not stated.\nWe agree with the reviewer. P(r) function plots for all the samples have been presented in\nSupplementary Figures 13, 14 and 19.\nQ3: In general, the SAXS results could be strengthened by including quantitative comparisons—\nfor example, reporting the Dmax: for wild-type TDP-43 (162 nm) alongside the range observed\nfor the mutants (159–165 nm). --> and quantify accordingly in line 286 as well the increase in\nDmax values (line 297) and later in text. Is there a way to estimate the errors from Dmax?\n(Obviously, the information is given in various tables, but could help the reader).\nWe thank to the reviewer. The program that we used to estimate the D is ATSAS-GNOM\nmax\n(shown in SAXS section in Materials and Methods). There is no error estimated for D that\nmax\nATSAS could calculate. D values have been added in the text (Page 7, lines 278-279 and Page\nmax\n8, lines 306-309).\nQ4: Elution profiles\n1. Supplementary Figure S9C -- Please comment on the shoulders - such as for the E17R mutants\nWe thank to the reviewer. The shoulder corresponds to an excess of free proteins. Indeed, as\nshown in the Supplementary File 1, an excess of protein than oligonucleotide target was used.\nMoreover, the absorbance value at 280 nm is higher than that at 260 nm (data not shown).\nSentences have been added to precise shoulders in the legend of Supplementary Figure 15 (Page\n49, lines 1750 and 1754).\nSupplementary Figure 11 -- Comment on the shoulders at frame 100 -- are these aggregates -\nhigher oligomer structures?\nWe thank to the reviewer. The shoulders at frame 100 indeed represents higher oligomeric\nstructures and not aggregates which eluate in the void volume of the column as shown for the\nprotein alone in Supplementary Figure 1. A sentence has been added to precise this point in the\nlegend of Supplementary Figure 18 (Page 55, lines 1973-1974). However, the signal has been\ndecomposed using the REGALS algorithm available in RAW as explained in the materials and\nmethods part (Page 71, lines 2519-2521).\nQ5: line 265---The chromatograms and the SAXS images were recorded during the elution. We\nobserved that the elution volumes were similar for all the conditions in the presence of (GT)12\nfor which the binding is cooperative.\" To improve clarity, consider explaining the underlying\nobservations in a bit more detail. E.g. The single mutants (blue and yellow trace) elute similarly\nas WT (red trace), which shows cooperative binding (see ITC Fig 1D, red plot)\nWe thank to the reviewer. This following sentence was added “In the presence of (GT) for\n12\nwhich the binding is cooperative (Figure 1D), we observed that the single mutants (blue and\nyellow trace) display similar elution volumes as WT (red trace) (supplementary Figure 13A).”\n(Page 7, lines 267-269).\nQ6: Supplementary Figure S9A -- I'm not sure what the difference is between the upper and\nlower panels. Perhaps a copy/paste issue with the legend?\nThe upper panel displays absorbance measured at 280 nm, while the lower panel displays\nabsorbance measured at 260 nm. It is now better indicated in the text (Page 44, lines 1591-1592).\nQ7: line 260 -- Restructure: To investigate structural changes on a larger scale than accessible by\nNMR, we performed SAXS analysis of in vitro–purified TDP-43–nucleic acid complexes.\nWe thank to the reviewer. The sentence has been restructured (Page 7, lines 261-262)\nQ8: line 264 -- The acquisition of the SAXS images WAS..... or SAXS measurements were\nperformed in-line with size-exclusion chromatography (SEC)\nWe thank to the reviewer. This mistake has been corrected (Page 7, line 265).\nQ9: line 265 -- specify which chromatgram (UV 280nm + 260nm ?)\nIn SAXS experiments, we used a combination of SAXS with on-line Size-Exclusion\nChromatography (SEC). The SAXS measurement is in-line after the UV measurement. The UV\nmodules can measure simultaneously the absorbance at 260 and 280 nm. SAXS images are\nrecorded just after the UV modules at a frames rate of 1 second per images. We precise SEC\nchromatogram in the text (Page 7, line 266).\nQ10: line 273 -- Already here, place the reference to Figure 3 and SAXS Table in supplementary\ninformation. (see line 277)\nWe agree with the reviewer. This point has been added in the text (Page 7, lines 274-275).\nQ11: line 274 -- As two hypotheses were formulated earlier be specific which one is in\nagreement.\nWe agree with the reviewer that it was not clear. The sentence has been changed clarify this\npoint (Page 7, lines 275-277).\nQ12: Line 1940 -- in Methods the use of REGALS for decomposing overlapping peaks is\nmentioned -- was this observed in the measurements?\nThe program REGALS was used only when we have the contamination of higher oligomeric\nstructures. The program REGALS was used to eliminate the impact of higher-order oligomer\ncontamination on the sample’s elution peak. Sentences have been added to explain this point in\nthe text (Page 71, lines 2519-2521 and Page 55, lines 1973-1974).\nQ13: Line 1945 -- not clear what is meant with: were acquired from averaged SAXS\nexperimental curves.\nWe calculate an average of several SAXS curves with a similar and stable Rg values to generate\na final SAXS curve allowing a better signal/noose ratio. A sentence has been added to clarify this\npoint (Page 71, line 2526-2527).\nQ14: Line 1950 -- models ... HAVE been generated\nWe thank to the reviewer. This mistake is corrected (Page 71, line 2538).\nGeneral:\n• To improve clarity and conciseness, consider introducing the E52R and R17E mutants, along\nwith the different nucleotide constructs, such as (GT)₆A₁₂(GT)₆, earlier in the text. This would\nreduce the need for repeated reintroductions later in the manuscript.\nWe agree. We have limited the number of reintroductions of E17R and R52E in the manuscript.\n• line 30 -- is it assembly of TDP-43 in (OR ON ?) introns\nWe agree. It has been changed into “on introns” (Page 1, line 31).\n• line 144 -- better: .. and includes the full NTD domain. (instead of while keeping)\nWe agree. The sentence has been changed into “including the full NTD domain” (Page 4, lines\n143-144).\n• line 148 -- ..we compared the chemical shifts of the RRM1-2 residues to contstucts containing\n(1-277) and lacking (101-277) the NTD domain\nWe agree. The sentence has been changed into “when the fragment contains or lacks the NTD\ndomain” (Page 4, line 148).\n• line 167 -- replace whatever with whether\nWe agree. It has been corrected (Page 4, line 165).\n• line 170 -- In THE presence of...\nThanks. “the” has been added in this sentence (Page 4, line 168).\n• line 172 -- remove 'a' to read: a major feature of cooperative association with (GT)₁₂\nWe agree. “a” has been removed (Page 4, line 170).\n• line 177 -- perhaps add 'red plot (Figure 1D)\nWe agree. “(red plot in Figure 1D)” has been added in the text (Page 5, lines 175-176).\n• line 178 -- why not mention the second mutant?\nWe agree. The second mutant E17R has been mentioned in the text (Page 5, line 179).\n• line 183 -- add \"green plot\"\nWe agree. “(green plot in Figure 1D)” has been added in the text (Page 5, lines 182-183).\n• line 214 -- 'A' similar result...\nWe agree. “The” has been replaced with “A” (Page 5, line 215).\n• line 267 -- add: the binding is cooperative (WT AND SINGLE MUTANTS).\nThe sentence has been changed into “In the presence of (GT) for which the binding is\n12\ncooperative (Figure 1D), we observed that single mutants (blue and yellow trace) display similar\nelution volumes as WT (red trace) (Supplementary Figure 13A).” (Page 7, lines 267-269).\n• line 290 -- remove 'The' before 'similar results'\nWe agree. “The” has been removed (Page 8, line 309).\n• line 317 -- replace 'To document' with to assess or investigate or characterise...\nWe agree. This point has been replaced (Page 8, line 336).\n• line 553 -- mutated TDP/43*aa. 1/277\nWe agree. “mutated” has been added (Page 15, line 601).\n• line 382 -- define 'HA'\nWe have defined HA-tag in the text (Page 74, lines 2619-2621)\n• line 401 -- use passive form : Two pairs of lysine residues, known to undergo acetylation\nfollowing arsenite stress, were mutated to arginine.\nWe thank to the reviewer. The sentence has been changed (Page 10, lines 429-430).\n• Supplementary Figure 2B: One could first show R52E mutant then the double mutant\nWe agree. The order has been changed in Supplementary Figure 4B.\n• Supplementary Figure 3: Color names should be in lowercase rather than uppercase.\nSorry. The mistake has been corrected in the legend of Supplementary Figure 6 (Page 35, lines\n1276-1277).\n• line 1089 -- interactions DO not interfere\nSorry. The mistake has been corrected (Page 34, line 1271).\n• line 1098 -- Significant CSPs ARE OBSERVED only in the binding residues of RRM1-2\n(assigned in black).\nWe agree. The description has been changed (Page 35, line 1280).\n• line 1099 -- COMPARED to ....\nWe agree. The description has been changed (Page 35, line 1281).\n• line 1142 -- Zoomed-in VIEW on THE chemical shift perturbations FOR TDP-43 residues in\nthe RRM, with or without the indicated nucleotides\nWe agree. The description has been changed into “Zoomed-in view on the chemical shift\nperturbations for TDP-43 residues in RRMs in the presence or absence of indicated\noligonucleotides” (Page 36, lines 1327-1328).\n• line 1144 -- R52E not R525E\nThanks. The mistake has been corrected (Page 36, line 1329).\n• line 1927 -- Data collection parameters ARE shown\nThanks. The mistake has been corrected (Page 71, line 2504).\n• line 1934 -- In each experiment -- (remove 's')\nThanks. The mistake has been corrected (Page 71, line 2512).\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for co-reviewing the manuscript.\nRevision of manuscript NCOMMS-25-35416B\n“From TDP-43/RNA complex formation to disease-linked TDP-43 aggregation through a\nstructural and cellular approach” by Yitian Feng et al.\nWe would like to thank the reviewers for their comments and constructive suggestions, which have\nsignificantly enhanced the quality and clarity of this manuscript.\nPoint-by-point response to reviewers’ comments\nReviewers’ comments and our answer (in blue):\nREVIEWERS' COMMENTS\nRemaining comments to address the previous concerns of reviewer #1:\n- Regarding reviewer #1’s concern regarding imaging parameters used, please clarify whether\nthere were differences in imaging parameters across experiments and why. This information\ncould be added to the results/methods.\nThere were no changes in imaging parameters when the same output was produced. Our pipelines\nare entirely automated. This comment is added to the materials and methods (See page 19).\n- Please comment on reviewer #1’s comments regarding the interchangeable use of 'granule'\nversus 'aggregate'. Please define this and update the main text.\nWe now indicated only “aggregates”. Granules is only mentioned for stress granules, which are\nreversible. A comment about the nature of the aggregates is indicated in page 10.\n- Regarding the rationale for not including data on the 6-point mutant and instead providing SEC\ndata comparing wt and mutant protein. The data does not show complete prevention of\ninteractions, but rather there there's at least a reduction. A trend for reduction is shown, but not\nproof of complete prevention. We kindly request these points are clarified, and the limitations of\nthese data are discussed in the text.\nWe show in supplementary figure 1 that a single mutant is largely enough to strongly limit NTD\nmultimerization by R52 mutation. First, SEC data combined to SAXS analysis which show clearly\nthat the size of R52E as well as E17R display a MW expected for a monomer form (Supplementary\nFigure 1). Second, from NMR data, the peaks of interface residues are displayed for R52E and\nE17R mutants contrary to WT form. Surprisingly, when we incubate R52E and E17R, we could\nreconstitute a WT behaviour with the disappearance of NMR peaks of interface residues\n(Supplementary Figure 9). Accordingly, we think that it is useless to perform 6-point mutations\nalong the NTD sequence covering several residues of interface (E14A, E17A, E21A, Q34A, R52A,\nand R55A) and consequently masks insights into the behaviour and dynamics of numerous\ninterface residues. Besides potential alterations in NTD structure, the presence of multiple\nmutations precludes us from carrying out dimer reconstitution using complementary mutations\n(Figure 2C, Figure 3B, Supplementary Figure 3, 6, 7, 9, 14). A comment was added (page 4).\nReviewer #2 (Remarks to the Author):\nI appreciate the authors efforts and their responsiveness to my concerns. I beleive all major\nconcerns have been addressed and the manuscript is much improved.\nOne small remaining concern, I had asked that the authors include the equations they used to fit\ntheir data, and while they did this with the SAXS analysis, they provided a link to the ITC\ninstrument's manual. I note that this document is on a password-protected site requiring\nregistration to access. Further, since this is a commercial site, the links and document may be\nupdated/revised/moved/discontinued etc thus not preserving critical information needed to\nreproduce the results of this paper. For full transparency and completeness, the authors should\ninclude the equations used to analyze the ITC data in the material and methods section. If length\nis a concern, then can be included in an expanded M&M section ion supplemental material.\nWe agree with the reviewer. The equations used for fitting the ITC data have been added in the\ntext (Pages 15-16, lines 604-620).\nReviewer #3 (Remarks to the Author):\nAll my comments have been addressed, and several figures have been added. In addition, some\ntext passages have been extended for greater clarity.\nI recommend that the manuscript be accepted for publication."}, {"round": 2, "reviewer_comments": {"reviewer_2": "I appreciate the authors efforts and their responsiveness to my concerns. I beleive all major concerns have been addressed and the manuscript is much improved.\nOne small remaining concern, I had asked that the authors include the equations they used to fit their data, and while they did this with the SAXS analysis, they provided a link to the ITC instrument's manual. I note that this document is on a password-protected site requiring registration to access. Further, since this is a commercial site, the links and document may be updated/revised/moved/discontinued etc thus not preserving critical information needed to reproduce the results of this paper. For full transparency and completeness, the authors should include the equations used to analyze the ITC data in the material and methods section. If length is a concern, then can be included in an expanded M&M section ion supplemental material.", "reviewer_3": "All my comments have been addressed, and several figures have been added. In addition, some text passages have been extended for greater clarity.\nI recommend that the manuscript be accepted for publication."}, "author_response": "We agree with the reviewer. The equations used for fitting the ITC data have been added in the\ntext (Pages 15-16, lines 604-620)."}]

Decision letter