Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct 30;133(20):jcs244129.
doi: 10.1242/jcs.244129.

TDP-43 dysfunction results in R-loop accumulation and DNA replication defects

Matthew Wood  1   2 , Annabel Quinet  1 , Yea-Lih Lin  3 , Albert A Davis  4 , Philippe Pasero  3 , Yuna M Ayala  5 , Alessandro Vindigni  6   2
Affiliations

TDP-43 dysfunction results in R-loop accumulation and DNA replication defects

Matthew Wood et al. J Cell Sci. .

Abstract

TAR DNA-binding protein 43 (TDP-43; also known as TARDBP) is an RNA-binding protein whose aggregation is a hallmark of the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43 loss increases DNA damage and compromises cell viability, but the actual function of TDP-43 in preventing genome instability remains unclear. Here, we show that loss of TDP-43 increases R-loop formation in a transcription-dependent manner and results in DNA replication stress. TDP-43 nucleic-acid-binding and self-assembly activities are important in inhibiting R-loop accumulation and preserving normal DNA replication. We also found that TDP-43 cytoplasmic aggregation impairs TDP-43 function in R-loop regulation. Furthermore, increased R-loop accumulation and DNA damage is observed in neurons upon loss of TDP-43. Together, our findings indicate that TDP-43 function and normal protein homeostasis are crucial in maintaining genomic stability through a co-transcriptional process that prevents aberrant R-loop accumulation. We propose that the increased R-loop formation and genomic instability associated with TDP-43 loss are linked to the pathogenesis of TDP-43 proteinopathies.This article has an associated First Person interview with the first author of the paper.

Keywords: DNA Replication; R-loops; RNA:DNA hybrids; TARDBP; TDP-43.

PubMed Disclaimer

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
TDP-43 knockdown induces DNA breaks and replication stress. (A) Neutral comet assay detecting DSBs in HeLa cells treated with control siRNA (siControl) or siRNA targeting TDP-43 (siTDP-43). Representative images of comets are shown. (B) Quantification of comet Olive moment. Data are represented as dot plot and means from five repeats, with mean values. n≥100 comets scored for each data set. ****P<0.0001 (unpaired t-test). (C) Schematic of the single-molecule DNA fiber tract labeling. HeLa cells transfected with siControl or siTDP-43 were sequentially labeled with IdU and CldU for 30 min each. Representative DNA fiber images after transfection with siControl or siTDP-43 are shown. (D) Size distribution of total tract length (IdU+CldU) in HeLa cells after transfection with control siRNA or siTDP-43 and siRNA-resistant WT FLAG-TDP-43 construct. HeLa cells treated with siControl or siTDP-43 were transfected with either 1 or 3 µg of WT FLAG-TDP43 vector. Data are pooled from three independent experiments and shown as dot plot. Bars represent the median of n≥150 tracts scored for each data set. ****P<0.0001; ns, non-significant (Kruskal–Wallis test with Dunn's multiple comparisons test). (E) Protein expression upon TDP-43 depletion (siTDP-43) and transfection with siRNA-resistant WT FLAG-TDP-43 construct. A representative western blot from three independent experiments is shown. (F) Representative immunofluorescence images of TDP-43 cellular localization after TDP-43 depletion and siRNA-resistant WT FLAG-TDP-43 vector expression. Scale bars: 5 µm (A,C), 10 µm (F).
Fig. 2.
Fig. 2.
TDP-43 knockdown induces genomic instability in neuroblastoma cells. (A) TDP-43 protein expression and the DNA damage marker γH2AX after knockdown with siTDP-43 in SH-SY5Y cells. Representative western blot (top) and quantification of γH2AX expression levels (bottom) after TDP-43 knockdown in SH-SY5Y cells. Data show fold increase in γH2AX expression compared with siControl-treated samples, from three independent western blots (mean±s.e.m.). *P=0.035 (paired t-test). (B) Quantification of Olive moments detected by neutral comet assay represented as a dot plot. Bars represent the mean of a total of five repeats; n≥100 comets scored for each data set. ****P<0.0001 (unpaired t-test). (C) Size distribution of total tract length (IdU+CldU) in SH-SY5Y cells after transfection with siControl or siTDP-43. Data are pooled from three independent experiments and shown as dot plot. Bars represent the median of n≥150 tracts scored for each data set. ****P<0.0001 (unpaired t-test). (D) Accumulation of chromosomal aberrations detected upon chromosome spread. Left: Representative images of chromosomes after SH-SY5Y transfection with siControl or siTDP-43. Red arrows point to the chromosome shown in the insert. A chromosomal break is highlighted in siTDP-43. Scale bars: 1 µm. Right: Percentage of cells with chromosomal abnormalities per data set (mean±s.e.m.); n≥50 metaphases scored for each of the three individual data sets. **P=0.0054 (paired t-test).
Fig. 3.
Fig. 3.
TDP-43 loss increases accumulation of R-loops. (A) Left: Representative immunofluorescence images of HeLa cells depleted for TDP-43 (siTDP-43) and treated with the RNase H nuclease probed with R-loop-specific (S9.6) and nucleolin antibodies. Right: Quantification of nuclear S9.6 intensity signal, minus nucleolar signal, shown as relative to siRNA control and untreated samples. n≥100 cells scored for each data set. Data are represented as mean±s.e.m. from four independent experiments. **P=0.0097; ns, non-significant (paired t-test). (B) Slot blot analysis of siRNA-treated HeLa nuclear fractions. Left: Representative image of 0.25 or 0.5 μg DNA loaded in duplicate and probed with S9.6. Samples (0.5 μg) were also treated with RNase H prior to loading. DNA (0.25 μg) was probed with dsDNA-specific antibody as loading control. The blot was probed with R-loop (S9.6) or dsDNA-specific antibodies. Right: Quantification of S9.6 signal in siTDP-43-treated samples in the presence and absence of RNase H relative to siControl using LI-COR Image Studio. Values show the S9.6 enrichment from 0.25 and 0.5 μg DNA loadings combined (n=7, mean±s.e.m.). *P=0.02, **P≤0.01 (mixed-effects statistical analysis with Geisser-Greenhouse correction; Tukey's multiple comparisons tests with individual variances computed for each comparison). (C) DRIP-qPCR analysis of HeLa cells treated with siControl or siTDP-43 with and without RNase H1 treatment. Left: The R-loop prone sequence SLC35B2 and an additional known negative control sequence SNRPN were probed. Data are represented as mean±s.e.m. from two independent experiments. **P=0.0062, ****P<0.0001; ns, non-significant (left) (two-way ANOVA with Holm-Sidak's multiple comparisons test, with a single pooled variance). Right: The R-loop prone sequence RPL13A and an additional known negative control sequence SNRPN were probed. Data are represented as mean±s.e.m. from three independent experiments. *P=0.0451, ***P=0.0008; ns, non-significant (two-way ANOVA with Holm-Sidak's multiple comparisons test, with a single pooled variance).
Fig. 4.
Fig. 4.
R-loop resolution by RNase H1 resolves replication stress in TDP-43 depleted cells. (A) Protein expression of TDP-43 and RNase H1 after TDP-43 knockdown and transfection with wild-type (WT) or nuclease mutant D145N RNase H1 constructs. A representative western blot from three experiments is shown. (B) Size distribution of total tract length (IdU+CldU) in HeLa cells after transfection with siControl or siTDP-43 and WT or nuclease mutant D145N RNase H1 constructs. Data are pooled from three independent experiments and shown as dot plot. Bars represent the median of n≥150 tracts scored for each data set. ****P<0.0001; ns, non-significant (Kruskal–Wallis test with Dunn's multiple comparisons test).
Fig. 5.
Fig. 5.
TDP-43-mediated replication stress is dependent on transcription. (A) Top: Schematic of the DNA fiber assay with the transcription inhibitor α-amanitin. α-Amanitin (5 µM) was added to the cell medium for 4 h prior to the DNA fiber assay and was kept in the medium throughout labeling with IdU and CldU thymidine analogs. Bottom: Size distribution of total tract length (IdU+CldU) in HeLa cells transfected with siControl or siTDP-43 and treated with α-amanitin. Data are pooled from three independent experiments and are shown as dot plot. Bars represent the median of n≥150 tracts scored for each data set. ****P<0.0001; ns, non-significant (Kruskal–Wallis test with Dunn's multiple comparisons test). (B) Top: TDP-43 protein expression after knockdown with siTDP-43 and α-amanitin treatment as detected by western blot. Bottom: Quantification of S9.6 intensity signal relative to siControl, untreated samples. n≥100 cells scored for each data set. Data are represented as mean±s.e.m. from five independent experiments. **P≤0.0088; ns, non-significant (repeated measure one-way ANOVA with Tukey's multiple comparisons test, with a single pooled variance).
Fig. 6.
Fig. 6.
Crucial TDP-43 domains in R-loop regulation. (A) Schematic of TDP-43 domains: N-terminal domain (NTD), nuclear localization sequence (NLS), RNA recognition motifs (RRM1 and RRM2) and C-terminal domain (CTD). The approximate positions of applicable mutations are indicated. (B) TDP-43 protein expression upon TDP-43 depletion (siTDP-43) and transfection of the siRNA-resistant TDP-43 mutants shown in A. Representative western blots from three independent experiments are shown. (C) Size distribution of total tract length (IdU+CldU) in HeLa cells after transfection with siControl or siTDP-43 and the siRNA resistant TDP-43 mutants. Data are pooled from three independent experiments and shown as dot plot. Bars represent the median of n≥150 tracts scored for each data set. ****P<0.0001 (Kruskal–Wallis test with Dunn's multiple comparisons test). (D) Quantification of S9.6 intensity signal in HeLa cells transfected with siTDP-43 and the siRNA-resistant TDP-43 mutants. n≥100 cells scored for each data set. Data are represented as mean±s.e.m. relative to control cells from five independent experiments. *P=0.002; **P=0.0018; ***P=0.0002; ns, non-significant (repeated measure one-way ANOVA with Tukey's multiple comparisons test, with a single pooled variance).
Fig. 7.
Fig. 7.
TDP-43 knockdown induces genomic instability throughout the cell cycle. (A) HeLa cells were transfected with siControl or siTDP-43 and ultrafine anaphase bridges (UFBs) were visualized using both anti-PICH and anti-BLM antibodies. Representative immunofluorescence images of UFB (left) and percentage of anaphase cells with UFBs (right) from three independent experiments (mean±s.e.m.). Dots represent the means of each independent repeat; n≥50 anaphase cells scored for each data set. *P=0.0261 (unpaired t-test). (B) siControl and siTDP-43-transfected HeLa cells were treated with cytochalasin B for 24 h to produce binucleated cells and were scored for micronuclei (red arrows) upon DNA staining with DAPI and cytoplasm staining using anti-β-tubulin antibody. Representative immunofluorescence images of micronuclei (left) and frequency of micronuclei per binucleated cell (right) represented as bar plot (mean±s.e.m.) from three independent repeats. Dots represent the means of each independent experiment; n≥100 binucleated cells scored for each data set. *P=0.0168 (paired t-test). (C) G1-specific 53BP1 nuclear bodies (NB) were scored in HeLa cells transfected with siControl or siTDP43 using anti-53BP1 and anti-cyclin A antibodies. Representative immunofluorescence images of 53BP1 foci formation in HeLa cells (left) and quantification of 53BP1 nuclear bodies per cyclin A-negative G1 phase cell (right) represented as bar plot (mean±s.e.m.) from three independent repeats. Dots represent the means of each independent repeat; n≥300 cyclin-A-negative cells scored for each data set. *P=0.0107 (paired t-test). Scale bars: 4 µm (A), 10 µm (B,C).
Fig. 8.
Fig. 8.
TDP-43 downregulation increases R-loops and DNA damage in primary neurons and proposed model. (A) Top: Representative immunofluorescence images of γH2AX staining in murine neuronal cells after transfection with siControl or siTDP-43. Bottom: Percentage of cells with ≥3 γH2AX foci. Data are represented as mean±s.e.m. from two independent experiments performed in triplicate, n≥100 cells scored for each data set. *P=0.0271 (paired t-test). (B) Top: Representative immunofluorescence staining images with the R-loop-specific antibody S9.6 in murine neuronal cells after transfection with siControl or siTDP-43. Nucleoli were labeled with anti-nucleolin antibody. Nucleolar S9.6 staining was subtracted before S9.6 measurement. Bottom: Quantification of S9.6 intensity signal shown as relative to siControl. n≥75 cells scored for each data set. Data are represented as mean±s.e.m. from two independent experiments performed in triplicate. ***P=0.0007; ns, non-significant (paired t-test). (C) TDP-43 regulates R-loop formation through a transcription-mediated mechanism. Under normal conditions (top), TDP-43 plays a role in preventing aberrant R-loop accumulation during transcription by associating with nascent RNA. Loss of TDP-43 nuclear function (middle) results in accumulation of R-loops that may be subject to DNA breaks (scissors), both of which are linked to DNA replication stress. Replication stress may include fork stalling or slowing, as indicated by STOP signs, and together with R-loop-associated DNA damage leads to an increase in DNA double-strand breaks (bottom). Scale bars: 5 µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abu Diab M., Mor-Shaked H., Cohen E., Cohen-Hadad Y., Ram O., Epsztejn-Litman S. and Eiges R. (2018). The G-rich repeats in FMR1 and C9orf72 loci are hotspots for local unpairing of DNA. Genetics 210, 1239-1252. 10.1534/genetics.118.301672 - DOI - PMC - PubMed
    1. Afroz T., Hock E.-M., Ernst P., Foglieni C., Jambeau M., Gilhespy L. A. B., Laferriere F., Maniecka Z., Plückthun A., Mittl P. et al. (2017). Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation. Nat. Commun. 8, 45 10.1038/s41467-017-00062-0 - DOI - PMC - PubMed
    1. Aguilera A. and García-Muse T. (2012). R loops: from transcription byproducts to threats to genome stability. Mol. Cell 46, 115-124. 10.1016/j.molcel.2012.04.009 - DOI - PubMed
    1. Amador-Ortiz C., Lin W.-L., Ahmed Z., Personett D., Davies P., Duara R., Graff-Radford N. R., Hutton M. L. and Dickson D. W. (2007). TDP-43 immunoreactivity in hippocampal sclerosis and Alzheimer's disease. Ann. Neurol. 61, 435-445. 10.1002/ana.21154 - DOI - PMC - PubMed
    1. Arai T., Hasegawa M., Akiyama H., Ikeda K., Nonaka T., Mori H., Mann D., Tsuchiya K., Yoshida M., Hashizume Y. et al. (2006). TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 351, 602-611. 10.1016/j.bbrc.2006.10.093 - DOI - PubMed

Publication types