Regulation of R‐Loop Dynamics by Proteins and Long Noncoding RNAs: An Emerging Paradigm for Cancer Treatment

ABSTRACT

R‐loops are three‐stranded nucleic acid structures comprising an RNA/DNA hybrid and a displaced single‐stranded DNA. While transient R‐loop formation is essential for various physiological processes, their persistent accumulation leads to genomic instability. Cancer cells exhibit elevated R‐loop levels due to hypertranscription, replication stress, and impaired DNA repair pathways. In this review, we provide a comprehensive overview of the molecular machinery that resolves R‐loops, including chromatin remodelers, transcriptional regulators, nucleases, and helicases. We also highlight the emerging roles of long noncoding RNAs (lncRNAs) in modulating R‐loop dynamics and explore how these RNA‐based mechanisms cooperate with canonical resolution pathways. Finally, we explore the potential of targeting R‐loop regulatory networks as a novel therapeutic strategy in cancer treatment.

R‐loops are three‐stranded nucleic acid structures whose dysregulation leads to genomic instability and cancer progression. This review summarizes the protein and lncRNA machineries that regulate R‐loop dynamics and discusses the therapeutic potential of targeting these pathways in cancer.

Abbreviations

ASO

antisense oligonucleotide

ATR

ataxia telangiectasia and Rad3 related

BAF

BRG1‐associated factor

BET

bromodomain and extraterminal domain

BRCA1

breast cancer type 1 susceptibility protein

BRG1

Brahma‐related gene 1

Chk1

checkpoint kinase 1

DDR

DNA damage response

DDS

drug delivery system

DDX5

DEAD‐box helicase 5

DHX9

DEAH‐box helicase 9

dMMR

deficient mismatch repair

FANCD2

Fanconi anemia group D2 protein

H3K36me3

histone H3 lysine 36 trimethylation

hnRNP

heterogeneous nuclear ribonucleoprotein

lncRNA

long noncoding RNA

MeCP2

methyl‐CpG‐binding protein 2

MEN1

multiple endocrine neoplasia type 1

MSI

microsatellite instability

MSI‐H

microsatellite instability‐high

PARP1

poly (ADP‐ribose) polymerase 1

PSIP1

PC4 and SFRS1 interacting protein 1

P‐TEFb

positive transcription elongation factor b

RNAPII

RNA polymerase II

SETX

senataxin

ssDNA

single‐stranded DNA

SWI/SNF

Switch/Sucrose Nonfermentable

TATDN2

TatD DNase domain containing 2

TERRA

telomeric repeat‐containing RNA

TKT

transketolase

TOP3B

topoisomerase IIIβ

TopBP1

topoisomerase IIβ‐binding protein 1

TopI

Topoisomerase I

TRC

transcription–replication conflict

XRN2

5′ → 3′ exoribonuclease 2

1. Introduction

R‐loops are three‐stranded nucleic acid structures formed during transcription, comprising an RNA/DNA hybrid and a displaced single‐stranded DNA (ssDNA), transiently formed during transcription [1]. While transient R‐loop formation is essential for various physiological processes, including immunoglobulin class switch recombination, DNA repair, Okazaki fragment maturation, transcriptional regulation, gene expression control, and telomere maintenance, their persistent accumulation of R‐loops leads to repair stress (RS), DNA double‐strand breaks, and consequent genetic mutation or recombination events [2]. Such genomic instability underlies the development of diverse diseases, including cancer, neurodegenerative disorders, and autoimmune conditions [3].

Under normal circumstances, nascent RNA dissociates from the DNA template to allow proper RNA processing. However, in certain genomic contexts, RNA re‐anneals to the template strand, forming a stable RNA/DNA hybrid and exposing the nontemplate strand as ssDNA [4] (Figure 1). R‐loops are estimated to occupy approximately 5%–10% of the human genome and vary in size from a few dozen to several kilobases [5]. Once considered mere by‐products of transcription, R‐loops are now recognized as functional structures that play critical roles in physiological processes. Nevertheless, their aberrant accumulation can severely compromise genome stability, highlighting the importance of tightly regulated R‐loop dynamics [4].

FIGURE 1.

Regulation of R‐loop dynamics by proteins and lncRNA regulators in cancer. R‐loops are three‐stranded nucleic acid structures composed of an RNA/DNA hybrid and a displaced single‐stranded DNA. R‐loop prevention is mediated by chromatin remodelers and transcriptional regulators, whereas excessive R‐loop accumulation can lead to DNA damage. R‐loop resolution is orchestrated by protein regulators such as nucleases, RNA helicases, and DNA repair factors, together with lncRNAs. R‐loop regulators represent potential therapeutic targets in cancer treatment, and BRD4 inhibitors, DHX9 inhibitors, and TUG1‐targeting antisense nucleic acid (ASO) are currently under clinical evaluation.

One complexity of R‐loop regulation in cancer cells is that, although the overabundance of R‐loops and RS is a consequence of tumor cell proliferation and is associated with tumor progression [6], cancer cells must paradoxically resolve excessive R‐loops and RS efficiently to circumvent severe replication failure and avoid replication catastrophe [7, 8, 9]. Cells employ multiple factors to prevent the harmful effects of unscheduled R‐loops. For instance, topoisomerases such as Topoisomerase I (TopI) relax DNA supercoiling to limit R‐loop accumulation [10], while RNase H removes the RNA strand of RNA/DNA hybrids in a sequence‐independent manner [11]. Several helicases, including PIF1, Senataxin (SETX), and FANCM, can unwind RNA/DNA hybrids in vitro [2] (Figure 1).

Recent studies have illuminated the intricate relationship between R‐loops and long noncoding RNAs (lncRNAs) in the context of cancer. lncRNAs exhibit a wide array of biological attributes, ranging from transcriptional regulation and chromatin modification to the modulation of DNA topology and interactions with other classes of RNA [12, 13]. Importantly, they have emerged as pivotal regulators of both R‐loop formation and resolution, thereby influencing gene expression and contributing to tumorigenesis. For example, specific lncRNAs can modulate the recruitment of RNA‐binding proteins and chromatin remodelers to R‐loop regions, ultimately affecting transcriptional elongation and DNA repair pathways [14, 15].

Understanding the complex interplay between R‐loops and lncRNAs provides novel insights into cancer biology and reveals potential therapeutic opportunities. Targeting R‐loop regulatory networks, including the modulation of lncRNA activity, may represent an innovative strategy to selectively induce DNA damage in cancer cells and enhance the efficacy of existing therapies. In this review, we summarize recent advances in the regulation of R‐loops and discuss the potential of exploiting R‐loop regulatory pathways as a novel therapeutic approach in cancer (Figure 1).

2. Fundamental Principles of R‐Loop Dynamics

2.1. Physiological Roles of R‐Loops

R‐loops contribute to a diverse array of physiological processes by acting as structural and regulatory intermediates. They play a role in transcription termination by promoting RNA polymerase II (RNAPII) pausing and facilitating release at gene ends [16]. In chromatin regulation, R‐loops recruit chromatin‐modifying complexes and help maintain CpG island hypomethylation [17]. They also play a role in DNA repair, particularly in processing double‐strand breaks [18]. In the immune system, R‐loops facilitate immunoglobulin class‐switch recombination by enabling programmed DNA cleavage events [19]. At telomeres, R‐loops formed by telomeric repeat‐containing RNA (TERRA) aid in telomerase recruitment, contributing to telomere length maintenance [20]. Furthermore, in mitochondria, R‐loops serve as replication intermediates that ensure faithful duplication of the mitochondrial genome [21]. These examples emphasize the broad functional significance of R‐loops in normal cellular physiology.

Cells employ multiple mechanisms to regulate R‐loop homeostasis (Figure 1). RNase H1 and H2, endonucleases specific to RNA/DNA hybrids, degrade the RNA strand within R‐loops [22]. In parallel, helicases such as senataxin (SETX), DEAH‐box helicase 9 (DHX9), and DEAD‐box helicase 5 (DDX5) displace the RNA and unwind secondary structures [3]. In addition, DNA repair‐associated proteins including breast cancer type 1 susceptibility protein (BRCA1) and poly (ADP‐ribose) polymerase 1 (PARP1) promote R‐loop resolution under stress conditions by facilitating helicase activity and recruiting repair factors [23, 24].

2.2. Pathological Consequences of Aberrant R‐Loop Accumulation

While R‐loops play essential physiological roles, their persistent accumulation has been implicated in a variety of pathological conditions, including cancer, neurodegeneration, and autoimmune diseases [3]. In these contexts, unresolved R‐loops can interfere with transcription elongation and DNA replication, leading to transcription–replication conflicts (TRCs) (Figure 2A), replication stress, and DNA double‐strand breaks [1, 2, 25].

FIGURE 2.

Aberrant R‐loop accumulation and its consequences in cancer cells. (A) Transcription–replication conflicts (TRCs) occur when the transcription machinery and the replication fork collide on the same DNA template, leading to replication stress, R‐loop accumulation, and DNA damage. (B) In cancer cells, aberrant oncogene activation increases RNA polymerase II activity, leading to excessive R‐loop formation. Replication stress further promotes R‐loop accumulation by inducing TRCs. Additionally, defective DNA repair mechanisms, such as BRCA1/2 loss, impair R‐loop resolution, stabilizing R‐loops. This aberrant accumulation of R‐loops triggers DNA damage, resulting in mutation accumulation and genomic instability. If damage persists, cells undergo apoptosis.

In cancer cells, R‐loop accumulation is significantly elevated due to oncogene‐induced hypertranscription, replication stress, and defective DNA repair [26] (Figure 2B). Transcription factors such as MYC and NF‐κB increase RNAPII activity and elongation rates, thereby disrupting RNA processing and promoting RNA/DNA hybrid formation [6]. Aberrant S‐phase progression and deregulated replication initiation frequently induce TRCs, further exacerbating R‐loop accumulation [27]. Additionally, BRCA1/2 mutations impair R‐loop resolution [28], while aberrant chromatin remodeling stabilizes RNA/DNA hybrids [29]. Defects in transcription, replication, and DNA repair collectively promote excessive R‐loop formation, which induces DNA damage and leads to mutation accumulation, contributing to cancer progression and therapeutic resistance [29, 30].

Notably, when R‐loop levels exceed the cellular tolerance threshold, they can trigger replication stress and apoptosis, thereby limiting cancer cell survival. To counteract this, cancer cells activate compensatory mechanisms for R‐loop control. These include chromatin remodeling factors, transcription regulators, RNA nucleases, and RNA helicases [22, 31, 32]. Recent findings highlight the contribution of long noncoding RNA (lncRNAs) as emerging components of these networks, cooperating with helicases to modulate R‐loop formation and removal [14]. This review focuses on the multifaceted mechanisms that govern R‐loop homeostasis in cancer, with particular attention to the lncRNA‐mediated regulatory machinery and its implications for genome stability and selective therapeutic targets (Figure 1).

3. Regulation of R‐Loops by Chromatin Remodeling Factors

Chromatin remodeling factors dynamically regulate transcription, replication, and DNA repair by utilizing ATP to alter nucleosome architecture. Among them, INO80 and Switch/Sucrose Nonfermentable (SWI/SNF) have been shown to play crucial roles in maintaining genomic stability by preventing R‐loop formation in cancer cells.

3.1. INO80 Complex: R‐Loop Remodeling and Resolution

INO80 is an ATP‐dependent chromatin remodeling complex that modulates nucleosome positioning to coordinate transcription and replication [33]. It is particularly involved in R‐loop remodeling and resolution to ensure smooth replication fork progression [34]. INO80 is specifically recruited to transcriptionally active genes and repeat‐rich genomic regions, where RNA–DNA hybrids are prone to accumulation. Here, it functions in concert with RNAPII to disrupt RNA/DNA hybrids, working alongside RNase H1 and other R‐loop removal factors such as SETX. Loss of INO80 leads to R‐loop overload, severely impairing replication fork progression and increasing replication stress, which ultimately induces double strand breaks. Additionally, the absence of INO80 disrupts the function of fork protection factors such as BRCA1 and Fanconi anemia group D2 protein (FANCD2), which safeguard stalled replication forks from degradation, thereby further accelerating genomic instability in cancer cells.

3.2. SWI/SNF Complex: Transcriptional R‐Loop Resolution and Chromatin Remodeling

The SWI/SNF complex is an ATP‐dependent chromatin remodeler that dynamically regulates nucleosome positioning to facilitate transcription factor access. This complex, which includes Brahma‐related gene 1 (BRG1) and BRG1‐associated factor (BAF) subunits, promotes RNAPII progression during both transcription initiation and elongation [35, 36]. Recent studies have revealed that SWI/SNF is also involved in resolving R‐loops that accumulate during transcription [37]. By facilitating RNAPII elongation, SWI/SNF prevents excessive RNA/DNA hybrid accumulation. Furthermore, BRG1 interacts with RNA helicase SETX and DNA repair factor FANCD2 to actively resolve R‐loops, ensuring proper transcriptional dynamics and genome stability.

4. Regulation of R‐Loops by Transcriptional Regulators

Transcriptional regulation and R‐loop formation are closely interconnected. Proper RNAPII elongation dynamics are essential for preventing persistent R‐loops, but disruptions in this process can have opposing effects. Some transcriptional regulators, such as BRD4 and PSIP1 (PC4 and SFRS1 interacting protein 1), facilitate RNAPII elongation, ensuring smooth transcriptional progression and reducing the likelihood of R‐loop formation [38, 39]. In contrast, multiple endocrine neoplasia type 1 (MEN1) acts as a negative regulator of RNAPII elongation, preventing excessive transcription speed that could disrupt co‐transcriptional RNA processing and promote R‐loop formation [40]. Together, these factors maintain the delicate balance necessary to prevent R‐loop‐induced genomic instability.

4.1. BRD4: Prevention of R‐Loop Accumulation Through Transcriptional Elongation

BRD4, a member of the bromodomain and extraterminal domain (BET) family, binds to acetylated histones to regulate transcription [41]. It prevents R‐loop accumulation by recruiting positive transcription elongation factor b (P‐TEFb), which facilitates RNAPII elongation [38]. This promotes continuous RNAPII movement, reducing transcriptional pausing and hybrid formation. Loss of BRD4 leads to abnormal RNAPII accumulation, increasing transcriptional stalling and promoting R‐loop formation. It also downregulates topoisomerase IIβ‐binding protein 1 (TopBP1), inactivating the ataxia telangiectasia and Rad3 related and checkpoint kinase 1 (ATR‐Chk1) pathway and impairing R‐loop‐induced DNA damage repair [38]. Consequently, cancer cells experience S‐phase apoptosis. BRD4 inhibitors have been developed over many years as anticancer agents due to the role of BRD4 in transcriptional regulation, and they are considered promising therapeutics in various malignancies. In recent years, the development of more selective and potent inhibitors, including PROTAC‐based degraders, has accelerated [42]. These agents suppress BRD4‐driven transcription and may also enhance R‐loop accumulation, increasing therapeutic vulnerability.

4.2. PSIP1: Chromatin Binding and R‐Loop Regulation

PSIP1 is a chromatin‐bound transcriptional regulator that recognizes histone H3 lysine 36 trimethylation (H3K36me3), a histone mark associated with active transcription [43, 44]. It facilitates RNAPII elongation at actively transcribed loci, thereby suppressing R‐loop formation. Additionally, PSIP1 contributes to DNA damage repair by interacting with PARP1, a key R‐loop processing factor [39]. Loss of PSIP1 leads to RNAPII stalling, increased R‐loop accumulation, and impaired PARP1‐mediated repair. This genomic instability renders PSIP1‐deficient cells particularly vulnerable to PARP1 inhibitors and genotoxic agents, highlighting PSIP1‐related pathways as potential therapeutic targets in cancer.

4.3. MEN1: Regulation of RNAPII Elongation and RNA Splicing

MEN1 fine‐tunes RNAPII elongation to ensure proper coordination with RNA splicing [40]. Transcription elongation is tightly coupled with co‐transcriptional RNA processing. Proper elongation kinetics facilitate efficient spliceosome assembly and accurate recognition of exon–intron boundaries. In contrast, excessively rapid elongation can disrupt this coordination, resulting in splicing defects such as exon skipping or intron retention. These aberrant transcripts are often not fully processed and can form RNA/DNA hybrids more readily [45, 46]. MEN1 acts as a molecular brake on elongation, maintaining the fidelity of co‐transcriptional splicing. Its loss accelerates RNAPII activity, resulting in widespread R‐loop accumulation [40].

5. Mechanisms of R‐Loop Resolution by RNA Nucleases

Recent studies have identified 5′ → 3′ exoribonuclease 2 (XRN2) and TatD DNase domain containing 2 (TATDN2) as critical players in R‐loop degradation in cancer cells. These nucleases prevent R‐loop persistence particularly in BRCA1‐mutant breast cancer and gliomas.

5.1. XRN2: Mediating R‐Loop Resolution Through RNF8 and TKT Pathways

XRN2, a 5′ → 3′ exoribonuclease, is a key regulator of R‐loop degradation, especially in transcription termination and genome maintenance [16, 47, 48]. Krishnan et al. demonstrated that RNF8, an E3 ubiquitin ligase, facilitates XRN2 recruitment to R‐loop‐enriched genomic regions through ubiquitination [49]. In BRCA1‐mutant breast cancer cells, unresolved R‐loops and TRCs are particularly pronounced. In these cells, the loss of RNF8 impairs the chromatin recruitment of XRN2, resulting in excessive R‐loop accumulation and ultimately inducing cancer cell death. These findings suggest that the RNF8‐XRN2 axis plays a critical role in maintaining genome stability in BRCA1‐deficient tumors.

A recent study by Fu et al. revealed that transketolase (TKT), a metabolic enzyme, interacts with XRN2 in response to chemotherapy, promoting R‐loop processing in glioma cells [50]. Under normal conditions, TKT is primarily cytoplasmic; however, chemotherapy agents such as temozolomide and cisplatin trigger its nuclear translocation. In the nucleus, TKT binds to XRN2, enhancing its ability to process R‐loops and maintain genomic stability. Loss of TKT leads to excessive R‐loops, exacerbating DNA damage and increasing chemosensitivity. This study highlights the TKT‐XRN2 pathway as a potential therapeutic target for overcoming chemotherapy resistance in gliomas.

5.2. TATDN2: A Dual‐Activity Nuclease in R‐Loop Resolution

TATDN2 has emerged as another key player in R‐loop processing. TATDN2 is a Mg²⁺‐dependent ribonuclease that selectively targets R‐loops for degradation [51, 52]. Unlike XRN2, which primarily functions as a 5′ → 3′ exonuclease, TATDN2 exhibits both exonuclease and endonuclease activities, suggesting a broader role in R‐loop resolution. TATDN2 is specifically enriched in R‐loop‐prone genomic regions, where it cleaves the RNA strand of RNA–DNA hybrids to facilitate hybrid dissolution [52]. In BRCA1‐deficient cancer cells, TATDN2 is essential for R‐loop clearance, and its depletion leads to increased R‐loop accumulation and DNA damage. The functional differences between XRN2 and TATDN2 suggest that XRN2 primarily resolves R‐loops post‐transcriptionally, particularly during transcription termination, whereas TATDN2 degrades persistent R‐loops at active transcription sites, including repetitive sequences and regulatory regions prone to hybrid accumulation.

6. Resolution of R‐Loops by DEAD/H‐Box RNA Helicases

RNA helicases are a family of ATP‐dependent enzymes that modulate RNA secondary structures and participate in various RNA‐related processes, including transcription, splicing, translation, and RNA degradation [53]. Among them, ATP‐dependent DEAD/H‐box RNA helicases play a pivotal role in R‐loop resolution [54, 55] (Table 1). These helicases are frequently overexpressed in cancer cells, where they contribute to genomic integrity [56]. This section focuses on three DEAD/H‐box helicases, DHX9, DDX5, and DDX47, and their distinct mechanisms in resolving R‐loops and preserving genomic stability in cancer cells.

TABLE 1.

Functional roles of DEAD/DEAH‐box RNA helicases in R‐loop resolution.

Protein	Role in R‐loop resolution	References
DDX1	Resolves R‐loops at DSBs to promote homologous recombination	Li et al. [57]
DDX3X	Physically interacts and stimulates the catalytic activity of RNaseH2	Secchi et al. [58]
DDX5	Facilitates R‐loop degradation by XRN2. Interacts with TOP3B in a pathway parallel to SETX. MCM8 recruits DDX5.	Mersaoui et al. [59]; Saha et al. [60]; Wen et al. [61]
DDX17	Binds and resolves R‐loops, Promotes replication fork restart via the MUS81‐LIG4‐ELL pathway.	Boleslavska et al. [62]
DDX18	Removes R‐loops at DNA damage sites and endogenous R‐loop‐prone regions. Interacts with PARP1 for recruitment to DNA damage sites.	Lin et al. [63]
DDX19	Resolves R‐loops via ATR‐Chk1 signaling, which phosphorylates DDX19 and promotes nuclear relocalization. Independent of its known mRNA export function.	Hodroj et al. [64]
DDX21	Resolves R‐loops upon RNAPII occupancy. Interacts with SIRT7 for deacetylation and activity enhancement. Recruits METTL3 for co‐transcriptional m6A modification.	Song et al. [65]; Hao et al. [66]
DDX23	Tumor suppressor frequently deleted in adenoid cystic carcinoma (ACC). Recruited to R‐loop sites through RNAPII pausing. SRPK2 phosphorylates DDX23, activating its for R‐loop resolution.	Sridhara et al. [67]
DDX39B	Unwinds R‐loops genome‐wide. Localized to active chromatin. Prevents R‐loop accumulation via THO complex and RNA processing factors.	Pérez‐Calero et al. [68]
DDX41	Tumor suppressor resolving R‐loops in active promoters. Prevents myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) by reducing R‐loop accumulation. Loss activates NF‐κB‐mediated inflammatory signaling.	Mosler et al. [69]; Weinreb et al. [70]
DDX47	Resolves R‐loops at highly transcribed loci and repetitive sequences. Directly unwinds RNA–DNA hybrids in vitro.	Marchena‐Cruz et al. [71]
DHX9	Resolves R‐loops to regulate transcription termination. Interacts with PARP1 to prevent R‐loop‐associated DNA damage. Recruited by TDRD3 to suppress promoter‐associated R‐loops. AKT1 recruits DHX9 under replication stress. Regulated by SUMOylation (K120) and ATR‐dependent phosphorylation (S321).	Cristini et al. [72]; Yuan et al. [73]; Liu et al. [74], Huang et al. [75]; Yang et al. [76]
DHX30	Resolves mitochondrial tRNA/DNA hybrids	Xu et al. [77]
DHX38	Safeguards zebrafish retinal development by preventing R‐loop accumulation.	Sun et al. [78]

6.1. DHX9: R‐Loop Resolution and Transcription Termination

DHX9 is an RNA helicase that directly binds to RNA/DNA hybrids and resolves R‐loops. Cristini et al. identified DHX9 as a major R‐loop‐interacting protein that is recruited to R‐loop‐prone regions to facilitate transcription termination [72]. DHX9 also associates with the DNA repair factor PARP1, preventing R‐loop‐induced DNA damage. Loss of DHX9 leads to excessive R‐loop formation and transcription termination defects, resulting in RNAPII stalling and an increased risk of TRCs. Moreover, the disruption of the DHX9–PARP1 interaction compromises DNA damage responses (DDRs), accelerating genomic instability in cancer cells. Recent findings have demonstrated that the DHX9 inhibitor ATX968, one of the most advanced inhibitors targeting R‐loop resolution factors to date, selectively induces R‐loop accumulation and replication stress in microsatellite instability‐high (MSI‐H)/deficient mismatch repair (dMMR) tumors, triggering apoptosis in cancer cells [79].

6.2. DDX5: Cooperation With TOP3B in R‐Loop Resolution

DDX5 is an RNA helicase involved in splicing and RNA processing, which also plays a crucial role in R‐loop resolution through its interaction with topoisomerase IIIβ (TOP3B). Saha et al. demonstrated that DDX5 and TOP3B cooperate to resolve R‐loops, preventing hybrid stabilization and genomic instability [60]. DDX5 facilitates TOP3B recruitment to R‐loop sites, where TOP3B enzymatically resolves R‐loops through a DNA–RNA decatenation mechanism. This process involves the cleavage of displaced ssDNA, followed by strand passage and untangling of the hybrid structure. DDX5 helicase further assists by unwinding RNA/DNA hybrids, ensuring proper transcriptional elongation and preventing R‐loop‐induced transcriptional stalling.

6.3. DDX47: ATP‐Dependent R‐Loop Resolution and Interaction With MeCP2

Unlike DHX9 and DDX5, DDX47 functions at R‐loop‐prone genomic regions, particularly in highly transcribed loci and repetitive sequences [71]. In addition to its direct helicase activity, DDX47 interacts with methyl‐CpG‐binding protein 2 (MeCP2), an epigenetic regulator that localizes to methylated CpG sites. MeCP2 facilitates the recruitment of DDX47 to genomic regions prone to R‐loop persistence. Cells lacking DDX47 exhibit a significant increase in R‐loops, along with enhanced DNA damage signaling, as indicated by γH2AX accumulation. Notably, DDX47 is frequently overexpressed in cancers with high MeCP2 levels, such as gliomas and breast cancer, suggesting a role in preventing aberrant R‐loops in transcriptionally active, MeCP2‐enriched genomic regions.

7. The Role of lncRNA TUG1 in R‐Loop Resolution and Its Therapeutic Implications

As discussed in previous sections, efficient R‐loop removal relies on the enzymatic activities of RNA nucleases and RNA helicases. Recently, lncRNAs are found as crucial regulators that support this process, revealing an additional layer of R‐loop regulation. TUG1 is a lncRNA that plays a role in tumor progression and treatment resistance in various cancer types by promoting malignancy through cell proliferation, apoptosis regulation, and epigenetic transcriptional control [80, 81, 82]. Suzuki and Iijima et al. found that TUG1 is rapidly upregulated under replication stress and facilitates R‐loop resolution, thereby reducing replication stress and supporting cancer cell proliferation [14] (Figure 3).

FIGURE 3.

TUG1‐driven R‐loop resolution promotes cancer cell proliferation. In cancer cells, replication forks frequently encounter R‐loops, leading to replication stress, fork instability, DNA damage, and prolonged S‐phase, ultimately triggering apoptosis. The lncRNA TUG1 is upregulated in response to replication stress and interacts with pRPA32 and the RNA helicase DHX9 to facilitate R‐loop resolution, particularly at microsatellite repeat regions, promoting cancer cell proliferation. Targeting TUG1 with ASO therapeutics disrupts this process, increasing DNA damage and apoptosis while sensitizing cancer cells to chemotherapy.

7.1. TUG1 Interacts With pRPA32 and DHX9 to Promote R‐Loop Resolution

TUG1 is transcriptionally induced by MYC and ATR–Chk1 signaling pathways, particularly under replication stress, and functions as a stress‐responsive lncRNA [14, 80]. Mechanistically, TUG1 facilitates R‐loop resolution by interacting with phosphorylated replication protein A 32 (pRPA32) and the RNA helicase DHX9 (Figure 3). These interactions appear to enhance the recruitment of R‐loop‐resolving complexes at sites of genomic instability. Furthermore, TUG1 dynamically expands its protein interaction network in response to genotoxic stress, engaging other DEAD/DEAH‐box RNA helicases, PARP1, heterogeneous nuclear ribonucleoproteins (hnRNPs), and topoisomerases suggesting a broader scaffolding function [83]. This activity is particularly pronounced at microsatellite repeat regions, where persistent R‐loops can promote microsatellite instability (MSI) [14, 84]. TUG1 depletion leads to enhanced R‐loop accumulation, replication stress, and apoptosis, underscoring its protective role in maintaining genomic integrity in cancer cells.

While TUG1 remains the only lncRNA with experimentally validated roles in R‐loop regulation in cancer, emerging evidence from other systems suggests that this may reflect a broader paradigm. For example, Lnc530, a stem cell–specific lncRNA identified in mouse embryonic stem cells, was shown to suppress R‐loop accumulation through cooperative interactions with DDX5 and TDP‐43 [15]. Although its relevance in cancer remains unknown, this finding supports the concept that lncRNAs may act as molecular scaffolds or guides for R‐loop‐resolving factors in a context‐dependent manner. Further research is needed to determine whether similar lncRNA‐based regulatory mechanisms exist across diverse cell types and disease states.

7.2. Implications for Cancer Therapy

Given its critical role in R‐loop regulation, targeting TUG1 represents a promising therapeutic strategy for cancer treatment. Notably, depletion of TUG1, which disrupts R‐loop resolution mediated by DHX9, markedly inhibits tumor growth, suggesting that strategies aimed at increasing R‐loop accumulation could provide an effective anticancer approach. Indeed, small‐molecule inhibitors of DHX9 have been developed [79], however, DHX9 knockout mice are embryonic lethal [85].

Although many chemotherapeutic agents increase genotoxic stress, cancer cells can adapt by acquiring replication stress tolerance, thereby developing resistance to treatment. In this context, TUG1 may facilitate R‐loop‐mediated stress adaptation, allowing cancer cells to withstand chemotherapy. Therefore, disrupting TUG1 function could enhance the efficacy of chemotherapeutic agents by exacerbating replication stress. Additionally, TUG1 interacts with topoisomerases (Top1, Top2A, and Top2B) [83], which function in R‐loop regulation. Given that tumors with DDR gene mutations are sensitive to topoisomerase inhibitors [86], DDR‐deficient tumors may also respond to TUG1‐targeted therapy. Accordingly, TUG1 expression may serve as a predictive biomarker of response to existing topoisomerase inhibitor therapies. Consistent with the therapeutic potential of TUG1 inhibition, our xenograft studies using glioblastoma cells demonstrated that systemic delivery of TUG1‐targeting antisense oligonucleotides (ASOs) via a drug delivery system (DDS) effectively suppressed tumor growth [14]. TUG1‐targeting ASO therapeutics are currently in clinical trials (JRCT ID: jRCT2041230136; https://jrct.mhlw.go.jp/en‐latest‐detail/jRCT2041230136), demonstrating the translational relevance of this discovery.

8. Protection of Stalled Replication Forks by RNA/DNA Hybrid Formation

While previous sections have focused on the deleterious effects of persistent R‐loops, recent findings suggest that RNA/DNA hybrids can also play protective roles under replication stress. Specifically, at stalled replication forks, transient hybrid formation helps maintain fork stability by preventing excessive nucleolytic degradation [2, 87].

Two recent studies by Song et al. and Xu et al. provided critical insights into this regulation [88, 89] (Figure 4). These studies demonstrated that RNAPII is recruited to stalled forks, where it forms RNA/DNA hybrids. These hybrids function as a protective barrier against excessive degradation by the nucleolytic enzyme DNA2, thereby preventing fork collapse and maintaining fork stability. The RNA helicase DDX39A subsequently resolves these hybrids in a timely manner, enabling controlled resection and fork restart. Xu et al. further showed that RAD51 facilitates DDX39A recruitment, linking R‐loop resolution to homologous recombination pathways [89]. Both studies also demonstrated that loss of DDX39A leads to excessive fork protection in BRCA1/2‐deficient cells, potentially contributing to chemoresistance [88, 89]. These findings highlight a context‐dependent, protective role of RNA/DNA hybrids in fork integrity and suggest therapeutic implications for replication‐stressed cancer cells.

FIGURE 4.

Regulation of RNA/DNA hybrids at reversed replication forks. Upon replication stress, stalled replication forks undergo fork reversal, forming a four‐way junction structure. When RNAPII is recruited to reversed forks, it transiently forms RNA/DNA hybrids, which serve as a protective barrier against excessive degradation by nucleases such as DNA2. The RNA helicase DDX39A resolves these hybrids at the appropriate timing, allowing controlled DNA2‐mediated resection and facilitating fork restart. In the absence of RNA/DNA hybrid formation, reversed forks become susceptible to degradation by nucleases, leading to fork instability and collapse.

9. Summary and Perspective

R‐loops represent a double‐edged sword in genome maintenance. While they have crucial physiological functions, their dysregulation can compromise genomic stability, particularly in cancer cells experiencing high transcriptional output and replication stress. Cells have evolved sophisticated mechanisms to regulate R‐loop dynamics, including chromatin remodeling factors, transcriptional regulators, nucleases, and helicases. The discovery of lncRNAs as regulators of R‐loop resolution suggests that genome stability is governed by a multilayered regulatory network rather than previously appreciated.

Despite significant advancements in understanding R‐loop biology, key questions remain. How do cells determine whether an R‐loop should be resolved or maintained for regulatory functions? What specific subsets of R‐loops require lncRNA‐mediated resolution? Moreover, how do these mechanisms contribute to cancer development and therapeutic resistance? Addressing these questions will be essential for the development of R‐loop‐targeted therapies.

Targeting R‐loop regulatory pathways offers a promising strategy for cancer treatment. Recent preclinical studies have demonstrated that modulating R‐loop levels can selectively impair tumor cell survival, particularly in cancers with DNA repair deficiencies. The ongoing development of nucleic acid therapeutics targeting lncRNAs such as TUG1 further supports the translational relevance of R‐loop regulation in oncology. Future research should focus on refining therapeutic strategies that exploit R‐loop vulnerabilities while minimizing off‐target effects.

In summary, the regulation of R‐loops is a dynamic and highly coordinated process. Understanding the precise mechanisms governing R‐loop homeostasis will provide critical insights into cancer biology and pave the way for novel therapeutic interventions.

Author Contributions

Miho M. Suzuki: writing – original draft. Keiko Shinjo: writing – review and editing. Tatsunori Nishimura: writing – review and editing. Yutaka Kondo: writing – review and editing.

Funding

This work was supported by the Japan Agency for Medical Research and Development [23ck0106816h0001 and 23ama221204h0002 to Y.K.] and the Takeda Science Foundation; and performed as research programs of the Grant‐in‐Aid for Scientific Research, the Japan Society for the Promotion of Science [23H02747 to Y.K., and 23 K06634 to M.M.S].

Ethics Statement

The authors have nothing to report.

Consent

The authors have nothing to report.

Conflicts of Interest

Y.K. is a current Editorial Board Member of Cancer Science. The other author has no conflicts of interest to declare.