Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage

Significance

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which progressive dysfunction of motor neurons leads to paralysis and death. There is currently no known pathway of disease pathogenesis. However, many genes with varying functions have been linked to familial and sporadic forms of ALS suggesting multiple possible disease mechanisms. One set of gene products, including ALS-linked FUS and TDP43, functions in transcription and RNA processing. We find that defects in transcription due to loss of function of FUS or TDP43 can lead to DNA damage, including in primary human neuronal cells, and hypothesize that dysfunction in their RNA processing roles leads to DNA damage in motor neurons that, if incompletely resolved, could contribute to motor neuron death and ALS.

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

Amyotrophic lateral sclerosis (ALS) is a disease of both upper and lower motor neuron dysfunction that leads to progressive paralysis and eventually death due to respiratory failure. No single model of ALS disease pathogenesis has been revealed; however, multiple disease-associated genes are known (1). The variation in function of these genes suggests that there may be multiple ALS molecular subtypes. That said, the familial ALS gene product list is also enriched in protein groups with related functions.

Mutations in multiple RNA processing genes, including FUS (FUS RNA-binding protein), TDP43 (TAR DNA-binding protein), SETX (senataxin), TAF15 (TATA box-binding protein-associated factor 15), EWSR1 (EWS RNA-binding protein 1), HNRNPA1 (heterogeneous nuclear ribonucleoprotein A1), and HNRNPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1), among others, give rise to familial ALS (fALS) (1). FUS and TDP43 are currently the best studied, given that mutations in these genes lead to classic histologic findings in ALS neural tissue and that similar histologic findings can be found in neural tissue of sporadic ALS cases (2). At autopsy, cytoplasmic TDP43 inclusions are found in ALS motor neurons from patients with (i) TDP43 mutations and (ii) sporadic ALS (i.e., patients with no FUS, TDP43, or other known pathogenic mutation) (2). They are also present in neurons in various parts of the brains of patients with either sporadic or familial versions of the ALS-related disorder, fronto-temporal lobar dementia (FTLD) (2). At autopsy, FUS inclusions were detected in the cytoplasm of motor neurons of FUS mutation-bearing fALS patients and in the cytoplasm of neurons in various regions of the brain in some FTLD patients (2).

FUS and TDP43 exhibit a wide range of functions, most of which involve transcription and RNA processing. However, the significance of their cytoplasmic inclusions in patients with sporadic and fALS is unclear with respect to disease pathogenesis. Conceivably, nuclear TDP43 and/or FUS undergo a toxic loss of function when the molecular pathways in which they participate are disrupted. By contrast, these cytoplasmic inclusions might also mirror a toxic gain of function, which, in turn, triggers motor neuron cell death.

Animal models and cell-based assays have illuminated certain functions of FUS and TDP43 in both cycling and terminally differentiated cells (3–5). Work on TDP43 has suggested roles for it in transcription regulation, mRNA splicing, mRNA transport, and stress granule formation among other functions (4). FUS also plays a role in multiple cellular processes including transcription regulation, mRNA splicing and transport, stress granule formation, homologous DNA pairing through its DNA binding domain, and various forms of DNA damage repair (5). More specifically, FUS participates in both homologous recombination (HR) and nonhomologous end joining (NHEJ)-directed DSB repair (6, 7). The evidence suggesting a role for FUS in DNA repair is particularly intriguing for ALS pathogenesis, given growing evidence that RNA binding proteins are active in the prevention and repair of transcription-associated DNA damage (8–12). Other ALS-related RNA-binding proteins like SETX, EWSR1, and TAF15 have also been linked to DNA damage and repair, further suggesting the importance of a breakdown in this process in ALS pathogenesis (11, 13–16).

Depletion of various RNA binding/processing proteins triggers γH2AX (gamma H2A histone family, member X) focus formation, a canonical marker of DNA damage (10). This result suggests that these proteins are important in preventing or repairing DNA damage associated with transcription and RNA processing (10). Transcription-driven DNA damage can arise from (i) collapse of transcription machinery and the subsequent evolution of double-strand DNA breaks (DSBs); (ii) collisions between transcription and replication machinery, again leading to DSBs; (iii) damage to genomic segments that have been opened up/prepared for transcription; and (iv) the formation and unnatural stabilization of DNA-RNA hybrid R loop structures (8–10). R loops comprise a segment of a transcribed DNA strand annealed to the nascent RNA transcript with the nontranscribed DNA strand looped out (8, 9). The latter can trigger the development of DNA damage (8, 9).

Motor neurons are terminally differentiated and quiescent. Thus, repair of DSBs cannot occur via HR and likely occurs, at least in part, via the more error prone process, NHEJ, which operates through much of the cell cycle (17). Accumulation of DNA damage in terminally differentiated motor neurons can eventually lead to motor neuron cell death and potentially ALS. Thus, any mechanism that leads to accumulating DNA damage, especially in G0 cells, is a candidate contributor to the pathogenesis of ALS.

Given these possibilities, we attempted to study the role of FUS and TDP43 in the prevention and/or repair of transcription-associated DNA damage. A better understanding of the nature of DNA damage associated with (i) a loss of FUS and TDP43 function and/or (ii) the role that these proteins play in preventing or repairing it, has the potential to generate insights into the pathogenesis of ALS.

Results

Depletion of FUS and TDP43 Leads to Excessive Transcription Stalling.

Arrest of transcription by the RNA Polymerase II (RNA Pol II) inhibitor, α-amanitin, triggers formation of γH2AX foci, a sign of DNA damage. If depletion of a protein leads to increased sensitivity to α-amanitin–induced transcription arrest, the protein of interest might be required for the prevention or repair of transcription arrest-driven DNA damage. Indeed, depletion of the tumor suppressor and DNA damage repair protein, BRCA1 (breast cancer 1), leads to increased α-amanitin sensitivity, and this effect is due to increased DNA damage (18). We detected multiple roles for BRCA1 in the prevention and repair of transcription-associated DNA damage, including in promoting the restart of transcription after UV arrest and preventing/repairing R loop-driven DNA damage (18). Thus, given their transcription/RNA processing association, we asked whether cells depleted of FUS or TDP43 exhibit an increase in α-amanitin sensitivity.

Specifically, we transfected cells with either a control siRNA (siGL2) or with two different siRNAs that targeted each protein (Fig. 1 A and D). Then sensitivity to α-amanitin was assessed in a colony-forming assay (Fig. 1 B, C, E, and F). The results show that depletion of FUS or TDP43 led to increased α-amanitin sensitivity, suggesting that loss of function of either protein leads to defective prevention or repair of transcription arrest-associated DNA damage.

Fig. 1.

Depletion of fALS proteins leads to increased sensitivity to a transcription stalling agent. (A) Western blot demonstrating that FUS siRNAs deplete FUS protein. (Top) Blot shows FUS protein levels in U2OS cells transfected with a control siRNA and the two FUS siRNAs that were also used in experiments depicted in B and C. (Bottom) This blot was stripped and stained with anti-tubulin antibody, which served as a loading control. (B) U2OS cells were transfected with the siRNAs used in A (the siGL2 control and two FUS-specific siRNAs), plated at a sufficient density for colony formation, and treated with varying doses of the transcription inhibiting agent α-amanitin for 24 h, at which point the media were removed and replaced with fresh non–drug-containing media. One week later, the cells were stained with crystal violet solution, and the colonies were counted. Dose–response curves and IC₅₀s were generated from these counts. The experiment was repeated three separate times for each siRNA. The average IC₅₀ from the three repetitions of the experiment for each siRNA is shown in the bar graph, with the error bars representing the SD between the IC₅₀s. The P values above the siFUS bars represent the significance of the difference between the siGL2 IC₅₀ and each siFUS IC₅₀ calculated using a paired, two-tailed t test in GraphPad Prism. (C) Average dose–response curves for the three repetitions of the colony-forming experiments described in B. The error bars at each dose represent the SEM between the three repetitions of the experiment for each siRNA. (D) Western blot demonstrating that the three TDP43 siRNAs each deplete TDP43 protein compared with the siGL2 (firefly luciferase) control in U2OS cells (Top). This blot was stripped and stained with tubulin as a loading control (Bottom). (E) U2OS cells were transfected with the siRNAs used in D and treated with α-amanitin as in B. Dose–response curves were generated from these results, and the data were analyzed as in experiments depicted in B and C. The P values above the siTDP43 bars represent the significance of the difference between the siGL2 IC50 and each siTDP43 IC₅₀ calculated using a paired, two-tailed t test in GraphPad Prism. (F) Average dose–response curves from the three replicates of the colony formation experiments described in E. Error bars at each dose represent the SEM between the three repetitions of the experiment performed with each siRNA.

To test whether the increased α-amanitin sensitivity was due to increased DNA damage, we performed alkaline comet assays on the above-noted FUS- and TDP43-depleted cells (Fig. S1). This assay was chosen because it can recognize both single and DSBs, each a possible outcome of transcription arrest. Depletion of either FUS or TDP43 in α-amanitin–treated cells led to increased DNA breakage, again implying that these proteins suppress or repair DNA damage associated with transcription arrest.

Fig. S1.

Depletion of fALS proteins leads to increased DNA damage accompanying transcription arrest. (A) Western blot demonstrating that the FUS and TDP43 siRNAs deplete each protein in the setting of H₂O or α-amanitin (AA) exposure. (Top) Blot depicts either FUS (Left) or TDP43 (Right) protein levels in U2OS cells transfected with a control siRNA and the two FUS or TDP43 siRNAs and then treated with H₂O or AA from one of the four repeat experiments averaged in C. (Bottom) These blots were stripped and stained with tubulin as a loading control. (B) U2OS cells were transfected with either a control siRNA (siGL2) or siRNAs targeting fALS genes of interest, treated with a toxic dose of α-amanitin or an equivalent amount of ddH₂O for 24 h and then analyzed by comet assay to determine the amount of DNA damage present. Tail length was used as a measure of DNA damage. Increased tail length corresponds to increased DNA damage. Representative photographs of U2OS cells transfected with siGL2 used with each treatment are shown here. The brightness and contrast were both increased by 40% in all photographs using PowerPoint. (C) Sixty pixels was set as a threshold for denoting a culture as demonstrating meaningful DNA damage, and the average percentage of cells with tail lengths greater (red) or less (blue) than 60 pixels for each siRNA with each treatment is shown from four separate experiments. The error bars represent the SD between the three experiments. The P value denoting the significance of the difference between the percentages greater than 60 pixels for H₂O and AA for each siRNA is denoted above each pairing (AA, α-amanitin).

FUS Localizes at Sites of Transcription-Associated DNA Damage.

The finding that FUS participates in the prevention or repair of transcription-associated DNA damage is consistent with the prior finding that FUS localizes to DSBs induced by a UV laser (6, 7, 19). The latter observation suggests two functional possibilities: (i) that FUS participates in the repair of DSBs, per se, and (ii) that it localizes to breaks located near sites of transcription-associated DNA damage. In keeping with the latter, FUS functions in both the soluble nuclear and chromatin fractions. In the chromatin fraction, it binds to the C-terminal domain of RNA Pol II and modulates its phosphorylation on Serine 2, which helps to regulate the transcription of certain genes (20, 21). FUS also colocalizes with active (serine 2 phosphorylated) RNA Pol II in undamaged cells (22). There it likely aids in the transcription of a subset of genes, given that altered RNA Pol II Serine 2 phosphorylation occurred when the protein bound to certain genes in fibroblasts from patients with FUS mutations (22). Given these results, it seems likely that FUS functions in the context of transcription-associated DNA damage.

To determine whether FUS localizes at sites of transcription-associated DNA damage, we searched for FUS at UV-induced DNA damage sites that are associated with transcription arrest (18, 23). UV exposure induces the formation of active RNA Pol II-containing nuclear foci that contain certain DNA damage markers such as phosphorylated-RPA (replication protein A), γH2AX, and BRCA1 (18). One possibility is that these foci are active sites of transcription-associated DNA damage repair.

Thus, we searched for FUS in post-UV RNA Pol II foci, using a methanol–acetic acid fixation method that reveals concentrated chromatin-based complexes, e.g., those that contain RNA Pol II (23). The underlying hypothesis was that FUS localizes at these sites as part of its role in transcription-associated DNA damage repair.

Using FUS and RNA Pol II antibodies that are target specific (Fig. S2), we were able to detect a significant increase in the number of UV-treated cells with colocalizing FUS and active RNA Pol II foci compared with untreated controls (Fig. 2A). This observation suggests that FUS localizes to sites of active transcription after UV damage (Fig. 2A).

Fig. S2.

Specificity of the antibodies used for detection of ALS proteins at sites of post-UV transcription-associated DNA damage responses. (A) An antibody that can detect the active form of RNA Polymerase II (RNA Pol II phospho-S5) was used to depict post-UV sites of transcription associated DNA damage. To assess the specificity of this antibody (labeled RPII in the photographs), the antibody was incubated with two different peptides before being used to stain U2OS cells. In one case, the antibody was incubated with an unphosphorylated peptide encoding the C-terminal domain (CTD) of RNA Pol II (first two columns), and in the second case the antibody was incubated with a peptide encoding the CTD of RNA Pol II phosphorylated on Serine 5 (last two columns). These mixtures were used to stain U2OS cells that had been exposed to either 0 J or 25 J and fixed with methanol:acetic acid 4 h later. Photographs of DAPI-RNAPolII costaining are shown on the left of each panel and of the RNAPolII staining alone on the right for each panel. DAPI is a nuclear marker. (B) U2OS cells were plated on coverslips on day 1, transfected with either a control siRNA (siGL2) or one of two different FUS-specific siRNAs (siFUS #7 or siFUS #8) on days 2 and 3, and fixed with methanol:acetic acid and stained for DAPI and FUS, using a mouse monoclonal FUS antibody (M-FUS) and a rabbit polyclonal FUS antibody (R-FUS), on day 4. A representative DAPI, M-FUS, and R-FUS photograph is shown for siGL2, siFUS-7, or siFUS-8. *Brightness was increased by 40% in every individual photograph in this figure using Microsoft PowerPoint. **These photographs are best viewed on a computer screen and not on a printed paper copy.

Fig. 2.

FUS localizes to sites of post-UV transcription-associated DNA damage. (A and B) U2OS cells were treated with 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for FUS and RNA Polymerase II phospho-S5 (RPII) (A) or FUS and γH2AX (B). The top row is the 0 J field, the middle row is the 4-h post-25 J field, and the bottom row is a magnified cell from the 4-h post-25 J field. Yellow arrows denote a cell in the 4-h post-25 J field in which there is colocalization that has been cut out and magnified in the next row. Bar graphs representing the percentage of cells that contain greater than or equal to three FUS/RPII and FUS/γH2AX colocalizing foci are located next to the row of magnified single cell photographs. Bars represent the average of three separate experiments, and error bars represent the SD between experiments. P values denoting the significance of the difference between the 0 J and 4-h post-25 J results are above the 4-h post-25 J bars. *Brightness was increased by 40% in every individual photograph in this figure using Microsoft PowerPoint. **These photographs are best viewed on a computer screen and not on a printed paper copy.

To test whether DNA damage was present at these sites, we costained for FUS and the DNA damage-associated proteins, γH2AX (Fig. 2B), phosphorylated RPA (Fig. S3A), and BRCA1 (Fig. S3B). A significant fraction of post-UV BRCA1-containing foci contained active RNA Pol II, γH2AX, and phosphorylated RPA, suggesting that a significant number of active RNA Pol II foci contain γH2AX and phosphorylated RPA and thus arose in association with transcription-associated DNA damage (18).

Fig. S3.

FUS localizes to sites of post-UV transcription-associated DNA damage with BRCA1. (A and B) U2OS cells were exposed to 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for FUS and phosphorylated RPA (pRPA) (A) and FUS and BRCA1 (B). The top row is the 0 J field; the middle row is the 4-h post-25 J field; and the bottom row is a magnified cell from the 4-h post-25 J field. Yellow arrows denote a cell in the 4-h post-25 J field in which there is colocalization. It was also isolated, magnified, and shown in its magnified state in the bottom row of A and B. Bar graphs representing the percentage of cells with greater than or equal to three colocalizing foci for each antibody pairing are also shown. Bars represent the average of three separate experiments; error bars represent the SD between experiments. P values denoting the significance of the difference between the 0 J and 4-h post-25 J results are depicted above the 4-h post-25 J bars. *Brightness was increased by 40% in every individual photograph in Microsoft PowerPoint. **These photographs are best viewed on a computer screen and not on a printed paper copy.

Here we detected increased post-UV nuclear colocalization of FUS with γH2AX (Fig. 2B), phosphorylated RPA (Fig. S3A), and BRCA1 (Fig. S3B), again suggesting that FUS localizes at transcription-associated DNA damage sites.

These results imply that, after UV damage-induced transcription arrest, FUS localizes at genomic sites where active transcription has been arrested by DNA damage, and it does so together with BRCA1 which is known to localize at such sites (18). Presumably, its role is to participate in the repair of this damage through its HR or NHEJ functions and/or to prevent the development of additional damage. FUS also colocalized with γH2AX (Fig. 2B), pRPA (Fig. S3A), and BRCA1 (Fig. S3B) in cells that were not ectopically damaged, suggesting that its baseline function in cycling cells includes some form of endogenous DNA damage prevention or repair.

TDP43 Localizes at Sites of Transcription-Associated DNA Damage Together with FUS.

We also asked whether TDP43 concentrates at transcription-associated DNA damage sites. Using a specific TDP43 antibody (Fig. S4), we tested whether TDP43 also colocalizes with active RNA Pol II after UV damage. Indeed, more cells revealed colocalizing TDP43 and active RNA Pol II foci after UV damage than in an undamaged state (Fig. 3A), suggesting that TDP43, like FUS and BRCA1, also localizes to sites of transcription associated DNA damage.

Fig. S4.

Antibody used for detection of TDP43. (A–C) U2OS cells were plated on coverslips on day 1, transfected with either a control siRNA (siGL2-A) or one of two different TDP43-specific siRNAs (siTDP43 #2-B or siTDP43 #3-C) on days 2 and 3, and fixed with methanol:acetic acid and stained for DAPI and TDP43 on day 4. A representative DAPI and TDP43 photograph is shown for siGL2, siTDP43-#2, or siTDP43-#3. *Brightness was increased by 40% in every individual photograph in Microsoft PowerPoint. **These photographs are best viewed on a computer screen and not on a printed paper copy.

Fig. 3.

TDP43 localizes to sites of post-UV transcription-associated DNA damage. (A and B) U2OS cells were exposed to 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for TDP43 and RNA Polymerase II phospho-S5 (RPII) (A) and TDP43 and γH2AX (B). The top row is the 0 J field; the middle row is the 4-h post-25 J field; and the bottom row is a magnified cell from the 4-h post-25 J field. Yellow arrows denote a cell in the 4-h post-25 J field in which there is colocalization that has been cut out and magnified. Bar graphs representing the percentage of cells with greater than or equal to three colocalizing foci for each antibody pairing are next to the row of magnified single cell photos. Bars represent the average of three separate experiments, and error bars represent the SD between experiments. P values denoting the significance of the difference between the 0 J and 4-h post-25 J results are depicted above the 4-h post-25 J bars. *Brightness was increased by 40% in every individual photograph in Microsoft PowerPoint. **These photographs are best viewed on a computer screen and not on a printed paper copy.

TDP43 also localized at sites of transcription-associated DNA damage, given its colocalization with γH2AX and phosphorylated RPA in an increased percentage of UV-damaged cells (Fig. 3B and Fig. S5A). Moreover, like FUS, TDP43 colocalized with γH2AX (Fig. 3B) and phosphorylated RPA (Fig. S5A) in undamaged cells, consistent with both proteins engaging in preventing or repairing spontaneously arising DNA damage.

Fig. S5.

TDP43 localizes to sites of post-UV transcription associated DNA damage and colocalizes with FUS. (A and B) U2OS cells were exposed to 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for TDP43 and pRPA (A) or TDP43 and FUS (B). The top row is the 0 J field; the middle row is the 4-h post-25 J field; and the bottom row is a magnified cell from the 4-h post-25 J field. Yellow arrows denote a cell in the 4-h post-25 J field in which there is colocalization that has been cut out and magnified. Bar graphs representing the percentage of cells with greater than or equal to three colocalizing foci for each antibody pairing are shown next to the row of magnified single cell photographs. Bars represent the average of three separate experiments, and error bars represent the SD between experiments. P values denoting the significance of the difference between the 0 J and 4-h post-25 J results are shown above the 4-h post-25 J bars. *Brightness was increased by 40% in every individual photograph in Microsoft PowerPoint. **These photographs are best viewed on a computer screen and not on a printed paper copy.

Finally, we tested whether FUS and TDP43 normally colocalize and observed that, in the absence of ectopically induced damage, FUS and TDP43 foci overlapped significantly (Fig. S5B). This result is consistent with the possibility that these structures are the products of certain common molecular events (Fig. S5B), as suggested by their known functional profiles (4, 5) and the results reported in Figs. 1–4. FUS and TDP43 colocalization also increased after UV treatment (Fig. S5B), which is consistent with the notion that they both participate, possibly even together, in dealing with transcription-associated DNA damage.

Fig. 4.

FUS and TDP43 are involved in the prevention or repair of R loop-associated DNA damage. (A) Representative images of U2OS cells cotransfected with siGL2 siRNA and either an empty vector (vec) or an RNASEH1-encoding plasmid (RNH) and stained for RNASEH1. (B) Extra coverslips from one of the four replicates of the RNASEH experiment not used for γH2AX assessment that were cotransfected with various siRNAs and empty vector (vec) were stained for FUS to assess depletion of FUS protein. Representative images are shown with DAPI on the left and FUS on the right. (C) Extra coverslips from one of the four replicates of the RNASEH experiment not used for γH2AX assessment cotransfected with various siRNAs and RNASEH1 (RNH) were stained for TDP43 to assess depletion of TDP43 protein. Representative images are shown with DAPI on the left and TDP43 on the right. (D) Dot plot representing the percentage of cell nuclei with greater than or equal to five γH2AX foci after cotransfection with various siRNAs and with either empty vector or RNASEH for each of four individual experiments (a minimum of 200 nuclei were counted in each experiment for each condition/siRNA). Each dot corresponds to a single experiment. P values assessing the significance of the difference between the vector and RNASEH1 value for each individual siRNA over the four replicates were calculated using a t test and are as follows: siGL2 = 0.248, siFUS #7 = 0.018, siFUS #8 = 0.010, siTDP43 #2 = 0.002, siTDP43 #3 = 0.029. *Brightness was increased by 20% in every individual photo in this figure using Microsoft PowerPoint. **These photographs are best viewed on a computer screen and not on a printed paper copy.

FUS and TDP43 Participate in Either the Prevention or Repair of R Loop-Associated DNA Damage.

A particularly interesting form of transcription-associated DNA damage is that associated with R loops, as described above. Multiple proteins participate in their resolution, including one protein linked to familial ALS, SETX (16, 24). When they are not resolved, e.g., due to loss of an R loop-resolving protein, unrepaired single-stranded breaks on the nontranscribed strand can evolve into DSBs and lead to cell death (16, 24, 25). The notion that FUS and/or TDP43 is involved in the prevention or repair of R loop-associated DNA damage is of interest because of the possibility that these proteins associate with DNA damage prevention or repair even in the setting of no exogenous damage (Figs. 2B and 3B). Moreover, other familial ALS proteins are involved in the prevention/repair of this type of damage (16, 24), and BRCA1, which colocalizes with FUS and likely TDP43 at post-UV RNA Pol II DNA damage foci, also participates in the prevention and/or repair of R loop-associated DNA damage (18, 26, 27).

Thus, we asked whether FUS and/or TDP43 participate in the prevention or repair of R loop-associated DNA damage by searching for a decrease in DNA damage following FUS or TDP43 depletion in cells that express ectopic RNASEH1. RNASEH1 specifically digests DNA-RNA hybrids such as those that give rise to R loops (25). Assessing DNA damage markers in the setting of gene-specific depletion and RNASEH1 overexpression has previously been used to determine whether proteins are involved in R loop-associated DNA damage prevention or repair (18, 28).

We assessed the results of RNASEH1 overexpression (Fig. 4A) together with FUS and TDP43 depletion or the transfection of an irrelevant siRNA, using an established immunofluorescence-associated fixation method (Fig. 4 B and C). We then costained cells for γH2AX, as well as for RNASEH1 expression. The enzyme was cotransfected with a control or FUS- or TDP43-specific siRNA.

After control siRNA transfection, there was no significant difference in the fraction of cells containing five or more γH2AX foci between vector and RNASEH1 transfected cells (Fig. 4D and Fig. S6). However, in multiple replicate experiments, there was an incomplete but statistically significant decrease in cells containing increased numbers of γH2AX foci in cells transfected with RNASEH1 compared with vector in both siFUS and siTDP43 transfected cells (Fig. 4D and Fig. S6). This result implies that part of the DNA damage confronted by both FUS and TDP43, like the other familial ALS protein SETX (16), was R loop associated.

Fig. S6.

FUS and TDP43 are involved in the prevention or repair of R loop-associated DNA damage. The bar graph shown here is a rerepresentation of the same data shown in Fig. 4 for ease of interpretation. It is a bar graph representing the percentage of cell nuclei with greater than or equal to five γH2AX foci after cotransfection with various siRNAs and with either empty vector (blue bars) or RNASEH1 (red bars). Each bar represents the average of four separate experiments (a minimum of 200 nuclei were counted in each experiment for each condition/siRNA), and error bars represent the SD between those experiments. P values above each siRNA pair denote the significance of the difference between the vector and RNASEH1 value for each individual siRNA and were calculated using a t test.

Motor Neurons Exhibit FUS Colocalization with UV-Induced RNA Polymerase Foci.

All of the experiments in Figs. 1–4 were performed in U2OS cells, which are cycling cells derived from an osteosarcoma. There are likely major differences in certain DNA repair responses (e.g., HR) between this rapidly cycling cancer cell line and quiescent motor neurons, the CNS cells affected in ALS. Therefore, it was important to test whether motor neurons behave similarly. This testing was accomplished using human cell line-derived motor neurons (29). Mature wild-type (WT) motor neurons were generated through directed differentiation as indicated by many neurons being MAP2 and Islet-1 positive (Fig. S7). MAP2 is a neuronal marker, and Islet-1 is a motor neuron-specific marker (30). Separate cell collections in wells derived from the same individual differentiation event were left unirradiated or were exposed to 25 J of UV-C and immunostained for FUS and either active RNA Pol II or γH2AX.

Fig. S7.

Motor neurons derived by directed differentiation. (A) Schematic of neuron differentiation. Motor neurons were produced through directed differentiation of stem cells using dual SMAD inhibition and additional small molecules to promote neurogenesis and ventralization of neurons. (B) Motor neurons were treated with 0 J and fixed as described and were stained for the motor neuron marker ISLET. A representative field of neurons is shown in the top two and bottom left panels, and a magnification of one neuron in the merge panel is shown at the bottom right. An arrow in the nonmagnified merge panel indicates which cells are shown in the magnified panel at the bottom. (C) Motor neurons were treated with 0 J and fixed as described and were stained for or RNA Polymerase II pS5 and the neuron marker MAP2. A representative field of neurons is shown in the top four panels, and a magnification of two neurons in the merge panel is shown at the bottom. An arrow in the nonmagnified merge panel indicates which cells are shown in the magnified panel at the bottom. *Brightness has been uniformly increased in every photograph using Adobe Photoshop. **These photographs are best viewed on a computer screen and not on a printed paper copy.

After immunostaining, observing red or green nuclear foci in these experiments indicates that these structures are enriched for FUS (red) or RNA Pol II/γH2AX (green), respectively. Yellow foci indicate colocalization of FUS with either RNA Pol II or γH2AX and imply a shared function/response. FUS colocalized with UV-induced RNA Pol II pS5 and γH2AX foci 4 h after UV treatment in some foci in some motor neuron nuclei in multiple experiments (Fig. 5 and Figs. S8–S11). Specifically, data from two individual experiments (representative images from both experiments in Fig. 5 and Figs. S8–S11) were pooled to reveal that exposure to UV light increased the percentage of foci with colocalization of RNA Pol II pS5 and FUS (51.2% of 404 cells, 4-h post-25 J vs. 7.6% of 499 cells, 0 J; P < 0.0001). Exposure to UV light also increased the percentage of foci with colocalization of FUS and γH2AX (68.5% of 295 cells, 4-h post-25 J vs. 38.6% of 365 cells, 0 J; P < 0.0001). This result implies that elements of FUS operation at sites of transcription-associated DNA damage are similar in G0 motor neurons, and, as shown above, in cycling cells. It also supports the view that this function occurs in motor neurons.

Fig. 5.

FUS localizes to sites of post-UV transcription-associated DNA damage in WT motor neurons. (A and B) Motor neurons were exposed to 0 J or 25 J, allowed to recover at 37 °C for 4 h, fixed with methanol:acetic acid, and costained for FUS and RNA Pol II pS5 (RPII) (A) or FUS and γH2AX (B). Images were obtained with a spinning disk confocal system at 100×. In each panel, the top row is the 0 J field, the middle row is the 4-h post-25 J field, and the bottom row is a magnified cell from the 4-h post-25 J field. Yellow arrows denote a cell in the 4-h post-25 J field in which there is significant albeit not complete colocalization. A single cell, denoted by the yellow arrow, was excised from the relevant photographs and magnified in the row of photographs below them. Bar graphs representing the percentage of cells that contain greater than or equal to one FUS/RPII or greater than or equal to three FUS/γH2AX colocalizing foci are located next to the row of magnified single cell photographs. Bars represent pooled data from two individual experiments. P values denoting the significance of the difference between the 0 J and 4-h post-25 J results are above the 4-h post-25 J bars. *Brightness has been uniformly increased in every photograph using Adobe Photoshop. **These photographs are best viewed on a computer screen and not in a printed paper copy.

Not every nuclear FUS focus colocalized with an RNA Pol II or γH2AX focus after UV damage. FUS has many functions, not all of which involve DNA damage (5), and FUS foci were detected in both UV-damaged and some untreated cells alike (Fig. 5 and Figs. S8–S11). Conceivably, in UV damaged cells, the FUS foci that did not colocalize with RNA Pol II or γH2AX might reflect its operation during stages of DNA repair before γH2AX concentration or at sites free of RNA Pol II or phospho-RNA Pol II. Alternatively, they may represent FUS foci wherein FUS is operating in a non-DNA damage-associated manner.

Discussion

The results of α-amanitin sensitivity, comet, RNASEH expression, and multiple DNA damage-linked immunofluorescence analyses repeatedly suggest that both FUS and TDP43 are necessary for the prevention or repair of transcription-associated DNA damage, e.g., in the form of R loop-induced disorder (Figs. 1–5). These findings are compatible with the hypothesis of a role for DNA damage prevention and/or repair in suppressing both familial and sporadic ALS. Consistent with this hypothesis, FUS colocalized with RNA Pol II pS5 at UV-induced DNA damage sites in human motor neurons (Fig. 5 and Figs. S8–S11).

Human ALS tissue studies and mouse models have not yet conclusively shown that markers of DNA damage coincide with signs of impending cell death in motor neurons (6, 31). However, these results do not rule out the possibility that unrepaired transcription-associated DNA damage contributes to motor neuron death in ALS. Based on work described here and that of others (5, 6), it is reasonable to propose a model of ALS pathogenesis in which various ALS proteins, such as FUS and TDP43, prevent or repair various forms of transcription-associated DNA damage e.g., R loop-associated damage. Perhaps, through the stable formation of hydrogel or fibrous states (32, 33), mutated ALS-related proteins with low complexity (LC) domains like FUS, TDP43, EWSR1, or TAF15 lose an important nuclear function and thereby fail to respond to transcription-associated DNA damage. This kind of damage is known to be associated with excessively stable R loops, stalled transcription machinery, and stable open DNA.

In postmitotic neurons, DSBs are repaired by error-prone NHEJ machinery and not by HR, which requires intact sister chromatids and hence DNA replication (34). Over time, these sites may accumulate mutations, genome rearrangements, and/or chromatid breaks. Enough genomic disorder might accumulate to result in or to accelerate cell death, the major feature of ALS. This genome disorder-induced cell death might be induced together with the coparticipation of other as yet undetected neurotoxic events (35). Determining whether some forms or stages of ALS are, at least in part, driven by ongoing transcription-associated DNA damage will require future work.

Based on the results described here and the work of others, there is already support for this view of ALS pathogenesis, especially with respect to FUS-related cases. Our results fit with earlier observations showing that FUS localizes to sites of UV-laser induced DSBs and supporting a role for FUS in DNA repair (6, 7, 19).

FUS is recruited to DSBs in proliferating cells where it forms a liquid phase compartment and where it is important in D loop formation in the initial phase of HR and in recruiting repair proteins to DSBs (6, 33, 36). In those studies, FUS localized to these linear arrays of DSBs in a PARP-dependent manner before the appearance of markers of DNA damage or proteins involved in the repair of DSBs (6, 7, 13). In addition, these repair phenomena decreased in the setting of FUS depletion, suggesting that FUS is necessary early on after damage has developed to stabilize damaged DNA, prepare it for repair, attract repair proteins, and/or induce modifications needed to recruit other repair proteins (6). The observation that FUS localizes at sites of transcription-associated DNA damage, colocalizes with markers of DNA damage in the absence of exogenously generated DNA damage, and participates in the prevention/repair of R loop-induced DNA damage fits with these prior findings. It is also consistent with the notion that FUS functions, at least in part, in response to transcription-associated DNA damage.

The finding that FUS localizes at sites of transcription-associated DNA damage after UV exposure suggests that it is recruited to DNA damage structures and once there, participates in the repair process directly, and/or marks or stabilizes the damaged structures for repair. Given existing observations from this work and others (6), FUS may arrive relatively early after damage has occurred at these sites, in part, to stabilize an “open” transcription structure, because (i) FUS forms liquid compartments at sites of DNA damage in cells (33) and (ii) without FUS recruitment, other DNA damage repair proteins, like NBS1 or Ku70, operate inefficiently (6). The fact that FUS did not colocalize with every RNA Pol II pS5 or γH2AX focus in a single U2OS or motor neuron nucleus (Figs. 2 and 5 and Figs. S8–S11) is not surprising. FUS exerts multiple nuclear functions (5). Thus, not every FUS focus in a cell need represent its participation in DNA repair. Alternatively, in UV-treated cells, where FUS was one of the earlier proteins to arrive, RNA Pol II pS5 or γH2AX foci may not yet have developed. In addition, it is conceivable that FUS marks sites of old, poorly repaired DNA damage that are no longer marked by γH2AX.

Furthermore, instead of being recruited, perhaps FUS is already present at certain chromatin sites before damage develops, having processed along the DNA together with the transcription machinery. Conceivably, such a process prepares it to deal with impending chromatin damage. Whatever the case, FUS is known to interact with active RNA Pol II and to regulate its phosphorylation (20–22). Moreover, its nuclear staining pattern and that of active RNA Pol II have been shown to overlap, in part, in unperturbed cell lines (20–22).

We also found that TDP43, like FUS, plays a role in transcription-associated DNA damage prevention and repair, in particular in R loop-associated DNA damage, and might share a role in stabilizing transcription-associated damage or aiding in its repair with FUS (Figs. 1, 3, and 4). In particular, TDP43 and FUS significantly colocalized in foci before exogenously generated DNA damage and colocalized even more so after DNA damage, suggesting the existence of certain shared functions for these proteins (Fig. S5B).

Some of these spontaneously arising foci in cells that were not exposed to exogenous DNA damaging agents also colocalized with γH2AX and pRPA, again suggesting that one of the baseline functions of these structures may be in endogenous DNA repair. It is possible that some of the other RNA binding proteins linked to ALS through genetic studies, including EWSR1 and TAF15, also play a role in one or more DNA repair processes and, when mutated, contribute to ALS development via a similar DNA damage-driven process (1).

A better understanding of the function of these RNA-binding and transcription/RNA processing-associated ALS proteins could also aid in identifying and generating new biomarkers of the disease that would help in making a definitive ALS diagnosis, in tracking disease progression, and ideally, in directing the application of mechanism-based therapies in the future.

Materials and Methods

A more complete description is available in SI Materials and Methods.

α-Amanitin Sensitivity Assay.

The assay was performed as described previously (18). Cells were plated on day 1, transfected on days 2 and 3, and plated at appropriate densities for colony formation on day 4. Enough cells to form between 100 and 300 colonies were plated in triplicate for each transfection at each dose. Four hours after plating, the media were changed to media containing various doses of α-amanitin (Sigma Cat. #A2263) for 24 h, at which point the cells were washed once with PBS, and fresh media containing no α-amanitin were added. The cells were grown at 37 °C until appropriate sized colonies had formed and then stained with crystal violet. Colonies were counted using a Microbiology International ProtoCOL colony counter. Statistical analysis was performed, as described in SI Materials and Methods, using GraphPad Prism software.

Comet Assays.

Cells were plated on day 1 and transfected with control or gene-specific siRNAs on days 2 and 3. On day 4, the medium was changed to medium containing 0.35 μM α-amanitin or an equivalent volume of water, and the cells were incubated in it for 24 h at 37 °C. On day 5, alkaline comet assays were performed on these cells using a Trevigen CometAssay Kit (VWR Cat. #95036-942) according to the manufacturer’s protocol. Analysis was performed as described in SI Materials and Methods.

Motor Neuron Differentiation and Immunofluorescence.

Motor neurons were derived from the Hues63 cell line based on a previously described protocol (29) modified to use adherent cells, without producing embryoid body precursors, and as follows. Stem cells were plated on matrigel (Corning) and cultured in mTeSR1 (Stemcell Technologies) until confluent. Then, the media was changed to neuron differentiation media: DMEM/F12:neurobasal 1:1 (Life Technologies) supplemented with nonessential amino acids, glutamax, B27 (Life Technologies), and N2 (Stemcell Technologies). From day 0 to day 6, neuron differentiation media was supplemented with 1 μM retinoic acid (Sigma Aldrich), 1 μM smoothened agonist (DNSK), 0.1 μM LDN-193189 (DNSK), and 10 μM SB-431542 (DNSK). From day 7 to day 14, neuron differentiation media was supplemented with 1 μM retinoic acid, 1 μM smoothened agonist, 4 μM SU-5402 (DNSK), and 5 μM DAPT (DNSK). Motor neurons were plated on poly-d-lysine and laminin-coated chamber slides (Corning) in neurobasal media supplemented with nonessential amino acids, glutamax, N2, B27, and neurotrophic factors (BDNF, CNTF, GDNF). After being plated, they were allowed to mature at least 2 wk before UV treatment and immunofluorescence staining. For Islet-1 staining in which no UV treatment was performed, motor neurons on chamber slides were fixed in 4% paraformaldehyde/PBS and processed for immunofluorescent staining with an Islet-1 antibody (Developmental Studies Hybridoma Bank #40.2D6).

UV Immunofluorescence.

For all experiments, cells were plated on coverslips on day 1. The fixation and staining method are briefly reviewed here (18, 23). UV treatment was performed as described previously (18). After various treatments or transfections, cells were washed with PBS, fixed in chilled methanol:acetic acid (3:1) at 4 °C for 15 min and then washed with PBS. Cells were exposed to blocking solution [1 mg/mL BSA, 5% (vol/vol) normal goat serum, 1% Triton X-100, and PBS] at 37 °C for 30 min and then washed in PBS. They were incubated in primary and then secondary antibodies diluted in blocking solution. Finally, cells were mounted with mounting medium containing DAPI (Vector Laboratories Cat. #H-1200) and then viewed and photographed. U2OS cells were photographed using a Zeiss fluorescent microscope. Motor neurons were photographed with the 100× objective of a spinning disk confocal from Yokogawa attached to a Nikon Ti inverted microscope. See SI Materials and Methods for details of statistical analysis.

RNASEH Assay.

This assay was performed as described previously (18, 28), and detailed methods are provided in SI Materials and Methods.

SI Materials and Methods

Cell Culture.

U2OS cells were grown in DMEM 10% (vol/vol) FBS/1% penicillin/streptomycin at 37 °C and 10% CO₂.

siRNA Sequences.

siRNA sequences include the following: siGL2 (Dharmacon Cat. #D-001100-01); siFUS #7 (Dharmacon Cat. #J-009497-07), GAUCAAUCCUCCAUGAGUA; siFUS #8 (Dharmacon Cat. #J-009497-08), GGACAGCAGAGUUACAGUG; siTDP43 #2 (Dharmacon Cat. #D-012394-02), GCUCAAGCAUGGAUUCUAA; and siTDP43 #3 (Dharmacon Cat. #D-012394-03), CAAUAGCAAUAGACAGUUA.

siRNA Transfection.

Cells were plated for transfection on day 1, transfected with 50 pmol each of various siRNAs using Lipofectamine RNAiMax Transfection Reagent (Life Technologies Cat. #13778150) on days 2 and 3, and assayed on day 4.

α-Amanitin Sensitivity Assay.

α-Amanitin (Sigma Cat. #A2263) was dissolved in filtered ddH₂O. Cells were plated on day 1, transfected on days 2 and 3, and plated at appropriate densities for colony formation on day 4. Enough cells to form between 100 and 300 colonies were plated in triplicate for each transfection at each dose. The cells were allowed to settle for 4 h at 37 °C, and the media were changed to media containing various doses of α-amanitin including 0 (equivalent volume of water to that used at the highest dose of α-amanitin was added to this media), 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, and 0.35 μM. Cells were incubated in media containing α-amanitin for 24 h at 37 °C. These media were then removed, the cells were washed once with PBS, fresh media containing no α-amanitin were added, and the cells were allowed to incubate at 37 °C until appropriate sized colonies had formed. Cells were then washed with PBS, stained with crystal violet solution, and dried. Colonies were counted using a Microbiology International ProtoCOL colony counter.

Statistical analysis was performed using GraphPad Prism software. The percentage of surviving cells compared with the 0 μM α-amanitin control for each dose and each siRNA was calculated for each individual replicate in each experiment. These values were entered into GraphPad Prism to generate a dose curve to which a nonlinear regression curve was fit for each individual siRNA in each experiment. The α-amanitin IC₅₀ was estimated for each siRNA in each experiment from this curve. The IC₅₀s calculated from all replicates for each siRNA were averaged, and the values are shown in the bar graphs in Fig. 1, with the error bars representing the SD between the replicates. The P values represent the significance of the differences between the IC₅₀s for the control siRNA and the individual gene-specific siRNAs, and these were calculated using a paired, two-tailed t test in GraphPad Prism. Dose curves representing the average value at each dose for all three replicates were also generated in GraphPad Prism. They are shown in Fig. 1.

Comet Assays.

Cells were plated on day 1 and transfected with control or gene-specific siRNAs on days 2 and 3. On day 4, the medium was changed to medium containing 0.35 μM α-amanitin or an equivalent volume of water, and the cells were incubated in it for 24 h at 37 °C. On day 5, alkaline comet assays were performed on these cells using a Trevigen CometAssay Kit (VWR Cat. #95036-942) according to the manufacturer’s protocol. Photographs of the comet tails were taken, and comet tail length was assessed using TriTek CometScore software. Four replicates of the experiment were performed for each siRNA. Each experiment included one observation in each experimental condition (water and α-amanitin for each siRNA), permitting analyses that preserve comparisons within a given experiment. The full experiment was then replicated. Data were therefore analyzed using a paired t test.

UV Immunofluorescence.

All immunofluorescent staining was viewed and later photographed using a Zeiss Axioskop 2 MOT microscope.

Primary and secondary antibodies included the following: mouse anti-BRCA1 clone SD123 (37), mouse anti-TDP43 (Abcam ab57105), mouse anti-FUS (Santa Cruz sc-47711), rabbit anti-FUS (Bethyl A300-302A), RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (Abcam ab5131), phospho RPA32 (S4/S8) (Bethyl Cat. #A300-245A), MAP2 (Novus 300-312), rabbit anti-γH2AX antibody (Abcam #ab2893), goat anti-rabbit IgG H&L (Alexa Fluor 488) (Abcam ab150077), goat anti-mouse IgG H&L (Alexa Fluor 594) (Abcam ab150116), and anti-chicken IgG Alexa Fluor 647 (Life Technologies #A21449).

For UV exposure, cells were plated on coverslips on day 1. On day 2, they were washed once with PBS, treated with either 25 J or exposed to air for an equivalent amount of time (0 J control), allowed to recover at 37 °C for 4 h, and then fixed in chilled methanol:acetic acid (3:1) at 4 °C for 15 min and washed with PBS. Cells were exposed to blocking solution [1 mg/mL BSA, 5% (vol/vol) normal goat serum, 1% Triton X-100, and PBS] at 37 °C for 30 min and then washed in PBS. They were then incubated in primary and then secondary antibodies diluted in blocking solution. Finally, cells were mounted with mounting medium containing DAPI (Vector Laboratories Cat. #H-1200), and then viewed and photographed.

UV treatment was performed using a 254-nm UV-C lamp (UVP Inc.), and dosage was measured using a UVX radiometer (UVP Inc.). The UV exposure experiments were repeated three separate times for each antibody pairing. For U2OS cells, nuclei with greater than or equal to three colocalizing foci were counted in the 0 J and 4-h post-25 J experiments, and the percentage of nuclei containing greater than or equal to three colocalizing foci compared with the total number of nuclei counted for the various antibody pairings was calculated for each experiment. The average percentage was calculated for the three separate experiments for each antibody pairing and is shown in the bar graphs in Figs. 2 and 3 and Figs. S3 and S5, with the error bars representing the SD between the three experiments. Each experiment included one observation in each experimental condition (0 J and 4-h post-25 J), permitting analyses that preserve comparisons within a given experiment. The full experiment was then replicated twice more. Data were therefore analyzed using a paired t test in Microsoft Excel.

For quantification of motor neuron results, nuclei containing more than or equal to one colocalizing FUS and RNA Pol II pS5 focus or greater than or equal to three colocalizing FUS and γH2AX foci were counted in the 0 J and 4-h post-25 J time points from two individual motor neuron derivation/DNA damage immunofluorescence experiments. The counts from these two experiments were pooled, and the percentage of nuclei containing greater than or equal to one colocalizing FUS/RNA Pol II pS5 focus or three colocalizing FUS/γH2AX foci compared with the total number of nuclei counted for the various antibody pairings was calculated. These percentages are shown in Fig. 5. The significance of the difference in FUS/RNA Pol II pS5 colocalization with or without UV exposure, and FUS/γH2AX colocalization with and without UV exposure was assessed using a χ² test; P values from this analysis are reported in Fig. 5 and the main text.

For siRNA-treated cells, untreated cells were plated on coverslips on day 1, transfected with various siRNAs on days 2 and 3, and then fixed and stained with appropriate antibodies.

For RNA-Polymerase II peptide competition experiments, cells were fixed and stained as described above. However, the primary antibody mixtures underwent an extra incubation step before being used for staining. The RNA polymerase II phospho S5 antibody was diluted in the blocking solution described above along with 2 μg of either RNA polymerase II CTD repeat YSPTSPS peptide-phospho S5 (Abcam ab18488) or the same unphosphorylated RNA polymerase II CTD repeat peptide (ab12795). These mixtures were incubated at 4 °C for 2 h while rotating end over end. The antibody mixtures were then used to stain cells, and secondary antibodies and mounting were performed.

Confocal Microscopy.

Confocal immunofluorescence images were collected using the 488- and 561-nm lasers with a Yokogawa CSU-X1 spinning disk confocal (Andor Technology) mounted on a Nikon Ti-E inverted microscope (Nikon Instruments). Images were acquired using a 100× Plan Apo objective lens with an Andor iXon 897 EMCCD camera (Andor Technology). Acquisition parameters, shutters, filter positions, and focus were controlled by Andor iQ software (Andor Technology).

RNASEH Assay.

This assay was performed as described previously (18, 28). U2OS cells were plated on coverslips on day 1, transfected with 50 pmol of various siRNAs on days 2 and 3, transfected on day 4 with either 4 μg of pcDNA3 empty vector or pcDNA3 RNASEH1 (25) using Lipofectamine 2000 (Life Technologies Cat. #11668019), and fixed for immunofluorescent staining 24 h later on day 5 using 3% paraformaldehyde. Cells were permeabilized using 0.5% Triton X-100 (0.5% Triton X-100, 20 mM Hepes, pH 7.4, 50 mM NaCl, 3 mM MgCl₂, and 300 mM sucrose dissolved in ddH₂O) and then costained for γH2AX (Millipore 05-636 at 1:500) and RNASEH1 (Proteintech #15606-1-AP at 1:300). Extra coverslips were stained with either FUS or TDP43 antibodies to assess depletion. Secondary antibodies were Abcam Alexa Fluor 488 anti-rabbit and 594 anti-mouse (Cat. #Ab150077 and Ab150116). Cells were photographed using a Zeiss Axioskop 2 MOT microscope, and the number of nuclei containing five or more γH2AX foci was counted for each siRNA with each treatment. The experiment was performed four separate times, and the average fraction of cells containing five or more γH2AX foci from each treatment was calculated from the results of all four experiments. For vector transfected cells, all DAPI-positive nuclei were counted, and the number of γH2AX foci in every nucleus was assessed. The number of DAPI-positive nuclei containing five or more γH2AX foci compared with the total number of DAPI-positive nuclei counted was used to calculate the percentage of cells with five or more γH2AX foci for the vector transfected cells. For the RNASEH1 transfected cells, only RNASEH1-positive nuclei were counted, and γH2AX foci were assessed in these nuclei. The number of RNASEH1-positive nuclei containing five or more γH2AX foci compared with the total number of RNASEH1-positive nuclei counted was used to calculate the percentage of cells with five or more γH2AX foci for the RNASEH1 transfected cells. A minimum of 200 cells were counted for each siRNA treatment in each experiment. Error bars were generated based on the SD between each experiment. Each experiment included one observation in each experimental condition (vector or RNASEH1 for each siRNA), permitting analyses that preserve comparisons within a given experiment. The full experiment was replicated four times. Data were therefore analyzed using a paired t test in Microsoft Excel.

Western Blots.

After each comet or α-amanitin sensitivity assay, there was a population of transfected cells that was not plated or used in the assay. These cells were pelleted and used for Western blot analysis to assess selected protein depletion phenomena for each of the replicates of the various experiments. Here cells were pelleted, washed with PBS, and lysed in NETN300 lysis buffer [300 mM NaCl, 50 mM Tris⋅HCl, pH 7.5, 1 mM EDTA, 0.5% Nonidet P-40, 10% (vol/vol) glycerol]. Protein concentrations of the lysates were assessed, and the lysates were then diluted to achieve equivalent concentrations using Laemmli running buffer containing β-mercaptoethanol. Equivalent amounts of each sample were loaded onto and electrophoresed through 4–12% Bis-Tris gels, transferred to 0.45-μm nitrocellulose membranes that were then blocked in 5% milk, and blotted with appropriate primary antibodies for at least 1 h or overnight at 4 °C. Blots were then incubated with appropriate HRP-conjugated secondary antibodies for 1 h, washed, and imaged using chemiluminescence reagent and film. Primary and secondary antibodies included mouse anti-TDP43 (Santa Cruz Cat. #sc-100871), mouse anti-FUS (Santa Cruz sc-47711), goat anti-mouse light chain HRP conjugate (EMD Millipore Cat. #AP200P), and mouse anti-tubulin (Sigma Cat. #T-5168).