- In silico and in vitro analyses of TDP-43 SUMOylation
Analysis of TDP-43 SUMOylation was first assessed in silico by using four different bioinformatic tools (JASSA, SUMO-plot, GPS-SUMO and SUMO-Hydro) which consistently predicted the Lys 136 residue and a hydrophobic SUMO-interacting motif (SIM3, 106-110 residues) as SUMOylation sites, suggesting both a Lys-mediated covalent and a SIM-mediated non-covalent binding of SUMO proteins to TDP-43 (Fig.1a). The two predicted SUMO binding sites are both located within the TDP-43 RRM1 domain, which is highly conserved along phylogenesis (Supplementary Fig.1a) and has an important function for target RNA recognition and binding.
Based on the in silico analyses, the SUMOylation state of the endogenous TDP-43 protein was then assessed in vitro by immunoprecipitation (IP) of human neuroblastoma SK-N-BE cell lysates with two different antibodies recognizing aminoacidic sequences at the N-terminal or at the C-terminal region of TDP-43. We found that a fraction of the endogenous TDP-43 protein was physiologically SUMOylated and that the SUMOylated form was more efficiently recovered using the C-terminal antibody, while the unmodified TDP-43 was similarly immunoprecipitated by both antibodies (Fig.1b). Similar results were obtained in HEK293T cells in which the SUMO-modified TDP-43 was preferentially recovered by the C-terminal antibody although both TDP-43 antibodies were able to immunoprecipitate the unmodified protein (Supplementary Fig.1b). These findings, together with the in silico analyses, suggest that a fraction of endogenous TDP-43 is likely to be SUMOylated at the N-terminal domain which is supposed to become less recognizable by the N-terminal antibody when modified by SUMO binding (Fig.1a).
We over-expressed SUMO-1 and UBC9 plasmids together or SENP1 construct in SK-N-BE cells to induce SUMOylation or de-SUMOylation, respectively. By IP assay with SUMO-1 antibody, we observed that the SUMOylated TDP-43 form increased in condition of SUMO-1/UBC9 over-expression, while SENP1 over-expression did not induce changes compared to control cells (Fig.1c). Moreover, the SUMO-1 antibody was able to recover TDP-43 also at its native molecular weight (Fig.1c), suggesting that TDP-43/SUMO-1 interaction may occur also through a non-covalent binding, consistent with the presence of the SIM3 region predicted in silico (Fig.1a). In line with these results, an increase of the covalently SUMO-modified TDP-43 was observed also in HEK293T cells upon SUMO-1 over-expression, while the unmodified TDP-43 form was similarly recovered by SUMO-1 antibody both in physiological condition and after induction of SUMOylation, confirming the non-covalent SUMO-1 binding to TDP-43 also in non-neuronal cells (Supplementary Fig.1c).
By subcellular fractionation assays we further assessed that the SUMO-modified TDP-43 protein was totally localized in the nucleus as shown by the specific band that disappeared when the N-Ethylmaleimide (NEM) reagent, used to inhibit protein de-SUMOylation, was omitted in the lysis buffer (Fig.1d).
- Characterization of the SUMOylation-resistant TDP-43 protein
Our in silico analyses and in vitro experiments suggested that TDP-43 protein is SUMO-1-modified, likely in the RRM1 domain, both covalently and non-covalently in neuronal-like and non-neuronal cell lines. To better study the role of covalent SUMO-1 binding to TDP-43, we generated a SUMOylation-resistant TDP-43 protein in which the putative Lys 136 was mutated to Arg (K136R). The K136R substitution is expected to have a negligible impact on the structure of the RRM1 domain, considering the similarity between Arg and Lys and the observation that Lys 136 is Arg in C. elegans. This was ascertained using Molecular Dynamics (MD) simulations, which showed an average root mean square deviation (RMSD) of 1.17 Å over the course of a 100-nanosecond MD run for the backbone atoms of the full RRM1 in K136R with respect to the WT system. This low value indicated that the K136R substitution did not disrupt the structural integrity of the RRM1 domain (Fig.2a). After computationally excluding a major impact of the K136R substitution on TDP-43 protein structure, we investigated the sub-cellular localization of the SUMOylation-resistant TDP-43 in SK-N-BE cells by immunofluorescence (IF) analysis and we observed that it was mainly localized in the nucleus, similarly to the wild-type protein (Fig.2b).
Next, we explored the RNA-binding properties of K136R, as the NMR structural ensemble and available X-ray crystallographic structures of RNA-bound TDP-43 RRMs showed that K136 itself establishes a number of important contacts with the target RNA and is therefore an important site for RNA-RRM1 interaction (Lukavsky et al., 2013; Kuo et al., 2014; Chiang et al., 2016). Indeed, the analyses of the 20 structures in the NMR ensemble showed that the side chain of K136 is not tied down to a buried conformation, but rather exposed and available for interacting with RNA (Supplementary Fig.2) or other possible substrates, as investigated in the present study. MD simulations showed that, in the SUMO-resistant variant K136R, the bulkier Arg residue is still flexible, exposed and able to maintain a number of direct contacts with the target RNA (Fig. 2c). To experimentally corroborate that the RNA-binding capability of the mutant TDP-43 K136R protein is broadly maintained, we performed UV-crosslinking immunoprecipitation (UV-CLIP) assays to test the binding activity of the SUMOylation-resistant TDP-43 protein to its RNA targets. To this purpose, we selected five RNA splicing targets containing both the canonical TDP-43 consensus binding sequence UGn (CFTR, MADD and TNIK genes) and the non-canonical one (POLDIP3 and STAG2 genes). Our UV-CLIP results showed that the mutant TDP-43 K136R bound to all RNA targets, although with a lower binding affinity compared to the wild-type protein (Fig.2d), in a condition where both exogenous proteins were immunoprecipitated with a similar efficiency in the assay (Supplementary Fig.3). Importantly, the TDP-43 ΔRRM1 protein, which is deleted of the RRM1 and was used as negative control in the assay, showed the total inability of binding to all the RNA targets analysed (Fig.2d). Finally, also the recombinant TDP-43 proteins carrying the ALS-associated mutations Q331K, M337V and A382T in the C-terminal domain were used to test their RNA-binding activity to the selected RNA targets. All these mutant TDP-43 proteins also showed a slight decrease of their RNA binding capacity compared to the wild-type protein (Fig.2e). Taken together, these observations indicate that the K136 residue is well exposed and possibly accessible for SUMOylation and that the SUMO-resistant K136R variant did not affect the folding of TDP-43 RRMs. Although showing a somewhat lower binding affinity, the K136R substitution did not disrupt the binding of target RNAs.
- Analysis of the splicing activity of the SUMOylation-resistant TDP-43 protein
We then investigated if the reduced RNA-binding capacity of the SUMOylation-resistant TDP-43 protein could compromise its splicing activity by performing minigene splicing assays in HEK293T cells knocked-down for TDP-43. By over-expressing siRNA-resistant wild-type or K136R TDP-43 constructs, we compared their ability to rescue splicing defects induced by endogenous TARDBP gene silencing. We used minigene plasmids expressing the splicing targets already tested in the UV-CLIP assays and including the target exons and the flanking intronic regions for CFTR (exon 9), MADD (exon 31), TNIK (exon 15) and STAG2 (exon 30b) (Fig.3a-d). Upon TARBDP gene silencing, the skipping of CFTR exon 9 (20.6%) and the inclusion of MADD exon 31 (89.7%) significantly decreased in comparison to siRNA-control condition (62.2% and 91.1%, respectively)(Fig.3a,b), as previously described [34, 48]. Although not statistically significant, the skipping of TNIK exon 15 (3.2%) and STAG2 exon 30b (2.3%) was also decreased compared to control cells (8.4% and 3.7% respectively)(Fig.3c,d) as already demonstrated (Colombrita et al., 2015; De Conti et al., 2015). When we over-expressed the siRNA-resistant wild-type TDP-43 protein, all the analysed splicing events were rescued with a splicing activity that, in line with an increased amount of TDP-43 production following transfection, was significantly higher (CFTR: 90.3%; MADD: 93.9%; TNIK: 64%; STAG2: 12.5%) with respect to the siRNA-control condition (Fig.3a-d). Upon TDP-43 K136R over-expression, all the analysed splicing events were still rescued compared to the siRNA-control sample. However, while MADD exon 31 inclusion was rescued by TDP-43 K136R similarly to the exogenous wild-type TDP-43 protein (93.8% and 93.9%, respectively; Fig.3b), the skipping of CFTR exon 9 (62.8%), TNIK exon 15 (27.5%), and STAG2 exon 30b (2.3%) were rescued to a lower extent (Fig.3a,c,d).
By further extending our splicing analysis to endogenous TDP-43 splicing targets, including MADD and STAG2 genes, we observed that, upon TARDBP gene silencing, TDP-43 K136R was as efficient as the recombinant wild-type protein in rescuing exon inclusion activity of MADD exon 31 (79.6% vs 79.9%), while it was less effective in the skipping activity on STAG2 exon 30b (65.7% vs 74.1%)(Fig.3e-g), confirming the results obtained in the minigene splicing assays (Fig.3c-d). To investigate if these results might depend specifically on the type of alternative splicing event regulated by TDP-43 (exon skipping vs exon inclusion), we also studied the well-known splicing target POLDIP3, containing a non-canonical recognition motif and whose exon 3 inclusion is promoted by TDP-43 [49, 50]. We observed that POLDIP3 exon 3 inclusion decreased to 29.8% after TARDBP gene silencing compared to the control condition (78%), as expected, but was rescued similarly by both the wild-type (42.5%) and the K136R (47.7%) TDP-43 proteins (Fig.3e,h).
Altogether our results show that the TDP-43 K136R protein has a less effective exon skipping activity, but retains an exon inclusion activity comparable to the wild-type protein in regulating target gene splicing. Moreover, this differential splicing activity occurs independently on the type of the consensus binding sequence present in the target intronic region (classical UGn sequence vs not classical recognition motif)(Fig.3i).
- TDP-43 SUMOylation and stress granules formation
Besides acting mainly as a splicing factor, TDP-43 is also implicated in cell response to stress and in stress granules (SG) formation in the cytoplasm . We previously showed that TDP-43 is able to form SG if its RNA-binding ability is preserved (Colombrita et al., 2009). Given the observed decreased binding affinity of the SUMO-mutant TDP-43 protein to its target RNAs (Fig.2d), we investigated its ability to be recruited into SG upon an acute oxidative stress stimulus. By analysing the sub-cellular distribution of TDP-43 in human neuroblastoma SK-N-BE cells exposed to sodium arsenite (0.5 mM) for 30 minutes, we observed that the TDP-43 K136R protein remained in the nucleus, while the wild-type TDP-43 formed cytoplasmic foci co-localizing with the SG marker TIAR, as expected (Fig.4a). When we analysed the mutant ALS-associated TDP-43 proteins (Q331K, M337V and A382T), we observed that they were all recruited into SG as the wild-type TDP-43 upon arsenite insult (Fig.4b).
These data suggest that SUMOylation may also regulate TDP-43 capability of responding to environmental insults and its recruitment into SG in the cytoplasm.
- TDP-43 nucleocytoplasmic trafficking after modulation of SUMOylation
To investigate the role of SUMOylation in the nucleocytoplasmic trafficking of TDP-43, we analysed the sub-cellular localization of the wild-type and the SUMOylation-resistant TDP-43 proteins upon modulation of SUMOylation by SUMO-1 or SENP1 over-expression in SK-N-BE cells. Both exogenous TDP-43 proteins distributed mainly in the nucleus in the mock-transfected cells, with 3.7% of TDP-43 WT-positive and 3.3% of TDP-43 K136R-positive cells also showing a diffused localization in the cytoplasm (Fig.5a,b). After induction of SUMOylation by YFP-SUMO1 over-expression, we observed no differences in the sub-cellular distribution of the two TDP-43 proteins compared to the control condition (3.9% TDP-43 WT-transfected and 2.8% TDP-43 K136R-transfected cells with TDP-43 protein also localized in the cytoplasm)(Fig.5a,b). On the other hand, when we induced de-SUMOylation by over-expressing YFP-SENP1 construct, we observed a significant increase of cells showing also a cytoplasmic distribution of the WT (19.3%) and K136R (9%) TDP-43 proteins, although the number of cells with the SUMOylation-resistant TDP-43 mislocalized in the cytoplasm was significantly lower compared to TDP-43 WT-expressing cells (Fig.5b).
Since our results showed that TDP-43 mislocalization in the cytoplasm increased upon promoting de-SUMOylation and it occurred at a lower extent for the SUMOylation-resistant TDP-43 protein, we then evaluated if such effect depended on the modulation of the NCT system or was specific for TDP-43. We considered two splicing factors, the TDP-43 interactor hnRNPA2B1 and NOVA1, which, like TDP-43, mainly localize in the nucleus, but also shuttle between the nucleus and the cytoplasm [53, 54]. In silico analysis did not predict any SUMOylation site nor SIM for hnRNPA2B1, while NOVA1 was highly predicted to have Lys 219 as a putative SUMO-binding site by two out of the three programs used (Supplementary Fig.4a,b). By IF assays we observed that, in control condition, recombinant NOVA1 prevalently localized in the nucleus with 16.4% of transfected cells showing both a nuclear and a cytoplasmic distribution of the protein (Supplementary Fig.4c,e). This percentage decreased, although not significantly, to 7.5% upon stimulation of SUMOylation by YFP-SUMO-1 over-expression, while it showed a trend to increase (19.9%) when YFP-SENP1 was over-expressed (Supplementary Fig.4e). Conversely, the sub-cellular localization of hnRNPA2B1 was not affected by either YFP-SUMO-1 or YFP-SENP1 over-expression and remained entirely nuclear in all the experimental conditions (Supplementary Fig.4d,f).
Our data indicate that the nucleocytoplasmic trafficking of TDP-43 and NOVA1 splicing factors is not regulated exclusively by the SUMO-modulation of the NCT system, but also depends on their intrinsic property of being SUMOylable targets.
- TDP-43 cytoplasmic mislocalization after treatment with the deSUMOylating TS-1 peptide
In order to confirm the effect of de-SUMOylation on TDP-43 molecular trafficking in physiological conditions, we treated SK-N-BE cells with the cell-permeable peptide TS-1, that we previously generated from the C-terminal domain of the SENP1 enzyme and proved to promote protein de-SUMOylation , and analysed the sub-cellular distribution of the endogenous TDP-43 protein. By western blot analysis we confirmed that 5 µM TS-1 applied for 4 hours was able to promote de-SUMOylation by inducing a significant decrease (0.42X) of the total amount of SUMOylated proteins compared to untreated cells and we found that TS-1 significantly decreased also TDP-43 protein content (0.75X)(Fig.6a,b). IF analysis revealed that TS-1 treatment caused changes also in SUMO-1 sub-cellular distribution because the proportion of cells showing both a nuclear and a cytoplasmic localization significantly increased from 21.75% to 37.25% in TS-1-treated cells (Fig.6c,d).
When we quantified TDP-43 sub-cellular distribution, the majority of SK-N-BE cells showed a nuclear localization of the protein before and after TS-1-treatment, but the percentage of cells showing also a TDP-43 cytoplasmic localization significantly increased from 20% in untreated to 25.5% in TS-1-treated cells (Fig.6c-d), indicating that TDP-43 trafficking can be modulated by the de-SUMOylating cell-permeable TS-1 peptide.
- Induction of SUMOylation by KCl and analysis of its effect on TDP-43 nucleocytoplasmic localization
To further study the effect of SUMOylation on TDP-43 trafficking, SK-NB-E cells were exposed to KCl stimulus (60 mM for 3 minutes), which is reported to up-regulate protein SUMOylation . We observed a significant increase (1.4X) of total protein SUMOylation compared to untreated cells, while TDP-43 content was unchanged (Fig.7a,b). By sub-cellular fractionation, we observed that KCl treatment induced a significant increase of total protein SUMOylation both in the nucleus (1.7X) and in the cytoplasm (1.5X) (Fig.7c,d). KCl treatment also caused an increase of the amount of cytoplasmic TDP-43 (2.6X)(Fig.7e) as well as of the SUMOylated TDP-43 form in the nucleus (1.6X)(Fig.7f), although total TDP-43 protein levels did not significantly change (Fig.7a,b,c). This result can be explained by the fact that both cytoplasmic TDP-43 and SUMO-TDP-43 represent only a minor fraction of total TDP-43 which is mostly nuclear and whose content may be not influenced significantly by such small variations.
By image analysis of TDP-43 sub-cellular distribution, we observed that KCl treatment induced a significant increase of the number of cells showing also a cytoplasmic localization of TDP-43 from 20.0% in physiological condition to 58.2% after exposure to KCl (Fig.7g,h). In line with these results, also the proportion of cells showing a nuclear and cytoplasmic localization of SUMO-1 increased from 21.7% to 55.5% upon KCl treatment (Fig.7h).
- TDP-43 pathological aggregation after treatment with the deSUMOylating TS-1 peptide
As the de-SUMOylating TS-1 peptide was shown to promote TDP-43 mislocalization in the cytoplasm and the process of TDP-43 aggregation is still poorly understood, we investigated the effect of TS-1 also on the distribution of the aggregation-prone TDP-43 C-terminal fragments (35KDa and 25KDa)(Fig.8a). We first evaluated the sub-cellular localization of the full-length GFP-TDP-43 protein, which was mainly distributed in the nucleus (74.9% of SK-N-BE cells) with 16% of transfected cells showing a diffused localization of the exogenous protein also in the cytoplasm and 9.1% of cells showing GFP-positive nuclear or cytoplasmic aggregates (Fig.8b,c). After 4h-treatment with the TS-1 peptide (5mM), we observed a significant decrease of the proportion of cells (67.7%) with only nuclear GFP-TDP-43 distribution and a significant increase of cells (24%) with also a cytoplasmic GFP-TDP-43 localization (Fig.8b,c). Conversely, TS-1 treatment induced no changes in the distribution of GFP-positive aggregates (7.9% of cells)(Fig.8c).
We then quantified the sub-cellular distribution of the GFP-TDP-35 protein, which lacks the first 89 N-terminal residues corresponding to the folded N-terminal domain , but includes part of the nuclear localization signal (NLS, 82-98 aa) and Lys136 (Fig.8a). In contrast to GFP-TDP-43, GFP-TDP-35 was always diffused both in the nucleus and cytoplasm of 44.2% of transfected cells or showed a cytoplasmic dotted distribution (puncta) in 25.1% of GFP-positive cells and cytoplasmic aggregates in 30.7% of cells (Fig.8b,d). Upon TS-1 treatment, the amount of cells with a nuclear and cytoplasmic distribution decreased to 33.8% and the percentage of cells with cytoplasmic GFP-TDP-35-positive puncta increased significantly to 33.7%, while the percentage of cells with cytoplasmic aggregates remained unchanged (32.4%)(Fig.8d).
When we analysed the GFP-TDP-25 C-terminal fragment, deleted of the NLS, RRM1 and part of the RRM2 domain (Fig.8a), it mainly formed aggregates, as previously reported , which were mostly distributed in the cytoplasm only (64.1% of transfected cells), but also both in the cytoplasm and in the nucleus (35.9% of GFP-positive cells)(Fig.8b,e). TS-1 treatment did not change the sub-cellular distribution of GFP-TDP-25 aggregates in the two compartments (67.2% cells with cytoplasmic-only aggregates and 32.8% cells with cytoplasmic and nuclear aggregates)(Fig.8e). To evaluate any possible effect of TS-1 treatment also on the formation of GFP-TDP-25 aggregates, we conducted a quantitative analysis of the number and size (area) of aggregates. We observed no differences either in the number of aggregates per cell (Fig.8f) or in their size arbitrarily assigned to four different size categories (<0.2 mm2, [0.2-0.5[ mm2, [0.5-1] mm2, >1 mm2)(Fig.8g) after cell exposure to the TS-1 peptide.
Finally, we investigated if GFP-TDP-35 and GFP-TDP-25 colocalized with the endogenous SUMO-1 protein. By IF analysis we observed that SUMO-1 largely colocalized with the nuclear GFP-TDP-25 aggregates, while GFP-TDP-43 and GFP-TDP-35 proteins or the cytoplasmic GFP-TDP-25 aggregates did not show any SUMO-1 colocalization (Fig.8h). Given these results, we reconsidered our previous quantitative analysis on GFP-TDP-25-transfected cells by filtering our data specifically for the nuclear-only GFP-TDP-25 aggregates. Nonetheless, also in this analysis, we found that neither the number (26.1 vs 21.6 per cell)(Fig.8i) nor the size (Fig.8l) of nuclear GFP-TDP-25 aggregates changed upon TS-1 treatment.