Looping forward: exploring R-loop processing and therapeutic potential

Abstract

Recently, there has been increasing interest in the complex relationship between transcription and genome stability, with specific attention directed toward the physiological significance of molecular structures known as R-loops. These structures arise when an RNA strand invades into the DNA duplex, and their formation is involved in a wide range of regulatory functions affecting gene expression, DNA repair processes or cell homeostasis. The persistent presence of R-loops, if not effectively removed, contributes to genome instability, underscoring the significance of the factors responsible for their resolution and modification. In this review, we provide a comprehensive overview of how R-loop processing can drive either a beneficial or a harmful outcome. Additionally, we explore the potential for manipulating such structures to devise rationalized therapeutic strategies targeting the aberrant accumulation of R-loops.

Keywords: DNA damage; DNA repair; R‐loops; genome instability; transcription.

Fig. 1

Functions of regulatory R‐loops. (A) R‐loops form at promoters and transcription termination sites and affect transcriptional programs by altering the histone/DNA methylation status or by recruiting chromatin remodelers or transcription factors. (B) R‐loops promote DNA repair by activating the DNA Damage Response, by recruiting protein factors that facilitate repair or by acting as repair intermediates. Pre‐existing or de novo synthesized RNAs (DDRNAs, dilncRNAs) form transient hybrids to guide DNA repair factors to the lesion, while RNA methyl‐modifications either direct the choice of repair pathway or stabilize them to promote repair. (C) Centromeric R‐loops are formed by long non‐coding RNAs transcribed from centromeres (cenRNAs) and facilitate chromosome segregation through an RPA‐dependent activation of ATR. Precise fine‐tuning of cen R‐loops is required for proper centromere assembly (CENPA recruitment) and DSB repair. (D) Telomeres are transcribed into telomere repeat‐containing RNA (TERRA), a G‐rich repetitive long non‐coding RNA which has the propensity for R‐loop formation, necessary for telomere maintenance. TERRA R‐loops are transient, cell cycle‐controlled and are regulated by the shelterin complex. Created with BioRender.com.

Fig. 2

R‐loops as a source of genome stability. R‐loops, if left unresolved, can be processed into single‐strand or double‐strand breaks, yet their impact extends to multiple DNA‐templated processes, further contributing to genome instability. (A) R‐loop formation leads to RNAPII stalling, DNA repair inhibition or replication stress. Persistent R‐loop accumulation introduces global gene expression changes and leads to truncated transcripts and the aggregation of aberrant proteins, further adding to transcription stress. (B) Replication forks encountering the transcription machinery in a head‐on (HO) or co‐directional (CD) orientation lead to distinct outcomes. HO conflicts, being more damaging than CD, create favorable conditions for R‐loop formation and DNA damage. (C) R‐loops impede DNA repair and inhibit end resection at DSBs, by physically blocking access to or by interfering with recruitment of repair factors. Created with BioRender.com.

Fig. 3

Unlocking the dual nature of R‐loops. R‐loops function as both regulators of gene expression, DNA repair, telomere maintenance or chromosome segregation, and as drivers of genome instability. This inherent dual nature of these structures is coordinated by RNA/DNA/chromatin processing factors that safeguard the genome from detrimental R‐loop‐instigated DNA damage or cellular dysfunction. The association of R‐loops with multiple diseases, including cancer, neurodegenerative or autoimmune disorders, underscores their clinical relevance. Therapeutic strategies targeting R‐loop manipulation show promise, with approaches ranging from promoting (e.g. fragile X syndrome) to inhibiting their resolution (e.g. cancer). However, the context‐dependent nature of R‐loops necessitates careful consideration to avoid disrupting cellular homeostasis. Future research aimed at understanding the intricacies of R‐loop biology and developing context‐specific interventions will be crucial for realizing their therapeutic potential while minimizing adverse effects. Created with BioRender.com.