Abstract
Purpose :
Nascent RNA can reinvade the negatively supercoiled DNA behind moving RNA polymerase and form a structure termed R-loops, where a ssDNA is accompanied by an RNA:DNA hybrid. mRNP biogenesis and export factors work in tandem to prevent R-loop accumulation and to warrant the maintenance of genome integrity. R-loops are frequently detected in replicating cells, can impair replication and lead to genomic instability. We wondered whether R-loops are a threat to genomic instability in non-replicating cells. We explore the existence and possible role of RNA-DNA hybrids in non-replicating post-mitotic retinal neurons.
Methods :
We used S9.6 antibody to detect RNA:DNA hybrid accumulation in retinal and brain sections by immunofluorescence microscopy, in presence and absence of RNaseH1 (a RNA:DNA hybrid specific ribonuclease). Retinal sections were also probed with antibodies against multiple proteins; involved in DNA repair and processing of R-loops. Findings were confirmed by chromatin immunoprecipitation followed by quantitative PCR. Affect of DNA double strand break (gamma irradiation) and Reactive Oxygen Species (H202) on DNA damage response and R-loop formation, was studied by analysing H2AX phosphorylation and 53BP1 foci formation.
Results :
S9.6 staining is observed in proximity to nuclear membrane, specifically in euchromatin region of Photoreceptor cells. S9.6 signal is not observed in brain cells and minimally in other retinal cell types. RNaseH1 was only localised to the inner segments of Photoreceptors and not intranuclear. Senataxin (RNA:DNA helicase), is highly expressed in Photoreceptor nucleii but not in other retinal neurons. Oxidative DNA damage (H2O2) frequently accumulates R-loops and result in genomic instability, as seen in cellular assays where RNaseH1 suppressed H2AX phosphorylation on H2O2 exposure.
Conclusions :
R-loops accumulate in post-mitotic photoreceptor cells and affect differentiation, cell survival and apoptosis signaling. Senataxin is associated with actively transcribed chromatin of Photoreceptor cells, indicating its helicase activity as preferential way to process R-loops in Photoreceptors. R-loop formation and oxidative DNA damage are interlinked. DNA repair proteins involved in sensing and processing of R-loop associated stress could be crucial for developing neuroprotective strategies for retinal degeneration.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.