R-loops—three-stranded nucleic acid structures comprising an RNA-DNA hybrid and displaced single-stranded DNA—are increasingly recognized as dynamic and reversible regulators of genome function with broad disease relevance. Once considered transcriptional byproducts, R-loops are now known to regulate gene expression, shape chromatin architecture, and coordinate DNA replication and repair. However, their pathological accumulation induces transcription-replication conflicts, DNA damage, and epigenetic reprogramming across cancer, neurodegeneration, autoimmunity, and skeletal disease. High-resolution mapping and functional genomics link R-loop formation to RNAPII transcriptional dynamics, local sequence features (such as GC enrichment and G4 structures), and topological tension, which are enriched at promoters, transcription start and termination sites (TSS/TTS), telomeres, and centromeres. Their stability and resolution are tightly regulated by topoisomerases, helicases, and RNases H1/H2. Furthermore, mitochondrial R-loops within the mitochondrial D-loop region couple energy metabolism with mitochondrial DNA replication. R-loop imbalance represents a convergent pathogenic mechanism. Disease-specific R-loop landscapes, together with profiles of regulatory protein expression and associated pathways such as the FA/ATRX/BRCA axis, hold promise for diagnosis, stratification, prognosis, and precision therapeutics. Emerging therapeutic strategies, including targeted delivery of RNase H1, CRISPR-based modulation, and biomaterial-assisted clearance, aim to restore genomic integrity and enable precision interventions. By repositioning R-loop as a functional regulator in genomic biology and disease pathogenesis, this study effectively bridges the gap between fundamental research and clinical translation. Among translational avenues, ATR inhibition in ALT-positive tumors and ATR-based approaches in spliceosome-mutant myelodysplastic syndromes represent the most immediate, trial-ready opportunities highlighted by our synthesis.
Zong et al. (Sun,) studied this question.