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

Matthew Wood,Albert A Davis,Alessandro Vindigni,Annabel Quinet,Yea-Lih Lin,Philippe Pasero,Yuna M Ayala

doi:10.1242/jcs.244129

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

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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Conclusion