Abstract

ABSTRACTTAR 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.

Highlights

  • R-loops are three-stranded nucleic acid structures that form during transcription when RNA annealing to the template DNA strand displaces the complementary DNA strand

  • In agreement with previous findings (Ayala et al, 2008a), we found that loss of TAR DNA-binding protein 43 (TDP-43) by siRNA-mediated knockdown (KD) in human HeLa cells led to increased levels of phosphorylated histone H2AX (Ser139, γH2AX), compared with TDP-43-proficient controls (Fig. S1A)

  • TDP-43 KD led to a defect in the incorporation of the thymidine analog 5-ethynyl-2′deoxyuridine (EdU) during S phase (Fig. S1B), suggesting that TDP43 loss perturbs DNA replication and promotes DNA damage accumulation, activating a G2 phase cell cycle arrest

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Summary

Introduction

R-loops are three-stranded nucleic acid structures that form during transcription when RNA annealing to the template DNA strand displaces the complementary DNA strand. R-loops play important physiological roles in several cellular processes, aberrant accumulation of R-loop structures can hamper DNA replication, repair and transcription and lead to genomic instability (Gan et al, 2011; Groh and Gromak, 2014; Huertas and Aguilera, 2003; Li and Manley, 2005; Tuduri et al, 2009; Wahba et al, 2011). The most common genetic link to familial and sporadic ALS and FTD is an intronic hexanucleotide repeat expansion (G4C2) in the C9Orf gene (C9-HRE), leading to increased R-loop formation and genomic instability in patient spinal cord tissue and animal models (Abu Diab et al, 2018; Haeusler et al, 2014; Walker et al, 2017) This collective evidence suggests that DNA damage and aberrant R-loop accumulation play a previously unappreciated role in ALS and FTD pathogenesis

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