Crosstalk Between R-Loops, RNA/DNA Modifications, and Cell Death Dynamics in Canine Models of Retinitis Pigmentosa

Purpose: Mutations in RPGRorf15 and PDE6B cause early-onset retinitis pigmentosa, yet the molecular factors driving photoreceptor degeneration remain poorly understood. Here, we characterize the pathological accumulation of RNA:DNA hybrids (R-loops) in two canine disease models-xlpra2/RPGRorf15 and rcd1/PDE6B-to provide new insights into disease pathogenesis. Methods: Archival canine retinal tissues were used for immunohistochemistry, TUNEL, Western blotting, and PCR. Outer nuclear layer regions were isolated via laser-capture microdissection to assess gene expression changes. Results: R-loop dynamics paralleled progression of apoptosis in both models. Most TUNEL-positive photoreceptor nuclei were also positive for R-loops and m⁶A RNA modification, suggesting potential crosstalk. Near peak apoptosis, colabeled nuclei accounted for ∼68% to 79% in xlpra2 (7-8 weeks old) and 59% to 78% in rcd1 (3-6 weeks old), although TUNEL-only nuclei were also present. At later stages (16 weeks), colabeling ranged from 76% to 100% in xlpra2 and 52% to 53% in rcd1. By 16 weeks, R-loop accumulation declined in both models, whereas γH2AX, a marker of early DNA double-strand breaks, increased. Downregulation of genes controlling R-loop metabolism was evident at 6 to 7 weeks, including CPSF6 and XRN2 (both models), as well as BRD4 and SF3B1 in xlpra2 and DHX9 in rcd1. These alterations may partly underlie the disease-associated splicing defects observed in RPGRorf15 pre-mRNA, encoded by the R-loop-prone ORF15 locus. Conclusions: Photoreceptor degeneration in xlpra2 and rcd1 is accompanied by overlapping molecular features, including early R-loop- and m⁶A-associated apoptosis with concurrent transcriptional alterations in R-loop-regulatory genes, followed by a progressively R-loop-independent DNA damage response.