Spliceosomal components protect embryonic neurons from R-loop-mediated DNA damage and apoptosis

Abstract

RNA splicing factors are essential for the viability of all eukaryotic cells; however, in metazoans some cell types are exquisitely sensitive to disruption of splicing factors. Neuronal cells represent one such cell type, and defects in RNA splicing factors can lead to neurodegenerative diseases. The basis for this tissue selectivity is not well understood owing to difficulties in analyzing the consequences of splicing factor defects in whole-animal systems. Here, we use zebrafish mutants to show that loss of spliceosomal components, including splicing factor 3b, subunit 1 (sf3b1), causes increased DNA double-strand breaks and apoptosis in embryonic neurons. Moreover, these mutants show a concomitant accumulation of R-loops, which are non-canonical nucleic acid structures that promote genomic instability. Dampening R-loop formation by conditional induction of ribonuclease H1 in sf3b1 mutants reduced neuronal DNA damage and apoptosis. These findings show that splicing factor dysfunction leads to R-loop accumulation and DNA damage that sensitizes embryonic neurons to apoptosis. Our results suggest that diseases associated with splicing factor mutations could be susceptible to treatments that modulate R-loop levels.

The roles of RNA in DNA double-strand break repair

“…Until recently, R-loops were considered to be a source of genome instability due to the increased risk of replication fork collapse, and exposure of the single-stranded non-template DNA to damaging agents. [91][92][93][94] However, R-loops have been shown to not directly cause genome instability 95 and are now being increasingly implicated in the repair of DNA damage and the preservation of genome stability. 17,68,96 Investigating R-loops and R-loop-interacting proteins RNase H selectively degrades the RNA component of an R-loop and is therefore a powerful tool in the investigation of these structures.…”

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

“…Much of the supporting evidence has relied on the use of the S9.6 mouse monoclonal antibody. S9.6 was initially reported to specifically recognize RNA:DNA hybrids [11], and has thus been widely used to isolate, sequence, measure, and image RNA:DNA hybrids in a variety of cell types from a variety of organisms [12][13][14][15][16][17][18].…”

R-Loops in Motor Neuron Diseases

“…Moreover, defining the roles of R-loops in motor neuron biology will be fundamental to understanding the selectivity of motor neuron death in neurodegenerative disorders. Indeed, Sorrels et al (2018) demonstrated that control of R-loop levels in neuronal cells during embryogenesis is crucial for the development of the central nervous system [95]. This information opens the possibility that R-loop-related motor neuron diseases have their pathogenic roots in early central nervous system development, which clearly could change many perspectives on these disorders.…”

“…Indeed, impaired mRNA splicing driven by loss of spliceosomal components promotes R-loop accumulation and DNA DSBs in neurons in a zebrafish model. Resolution of R-loops after RNase H1 overexpression rescued DNA damage and neuronal apoptosis in zebrafish splicing factor mutants [95]. The widespread splicing alterations that are induced by reductions in SMN protein levels in SMA are potentially associated with impaired formation of transcriptional R-loops and subsequent global DNA damage and stress responses, which could ultimately compromise motor neurons survival.…”