Pathophysiological Role and Diagnostic Potential of R-Loops in Cancer and Beyond
Abstract
:1. Introduction
2. Roles of Regulatory R-Loops in Gene Regulation and Genome Stability
3. Role of Unscheduled R-Loops as a Source of DNA Damage and Genomic Instability
4. Role of R-Loop-Binding Proteins and (Co-)Transcriptional Mechanisms in R-Loop Formation, Resolution, and Prevention of Aberrant R-Loop Accumulation
Protein | Function |
---|---|
Transcription initiation and capping | |
Capping enzyme-Pol II complex [62] | Responsible for transcription initiation by modulating displacement of nascent RNA during transcription, thereby promoting R-loop formation |
Transcription elongation | |
Facilitates Chromatin Transcription (FACT) complex [17] | Helps in preventing R-loop accumulation-causing TRCs |
Transcription termination, cleavage, and polyadenylation | |
Cleavage and Polyadenylation (CPA) factors [15,29,69] (PCF11, CLP1, FIP1L1, CFT2, WDR33) | Suppresses R-loop formation and facilitates efficient mRNA cleavage, thereby preventing replication-stress-associated DNA damage |
RNA processing and export | |
Transcription and export complex (THO/TREX complex; Tho2/THOC2, Hpr1/THOC1, Mft1, Thp2, Sub2/UAP56) [13,73] | Inhibits aberrant R-loop formation and transcription-associated recombination |
Splicing | |
Serine- And Arginine-Rich Splicing Factor SRSF2 [16] | Prevents the formation of mutagenic R loop structures |
RNA-Binding Protein With Serine Rich Domain 1 (RNPS1) [16] | Forms complex with ASF/SF2 to prevent transcriptional R-loops |
R-loop degradation | |
RNase H1/2 [10,74] | Prevents aberrant R-loop formation by timely removal of these hybrids |
R-Loop-processing factors (DNA–RNA helicases) | |
Senataxin (SETX) [75] | Binds to replication forks to protect its integrity across RNA-Polymerase-II-transcribed gene and unwinds unnecessary R-loops |
Aquarius (AQR) [76] | Prevents R-loop formation by unwinding DNA–RNA hybrids |
DExH-Box Helicase 9 (DHX9) [77] | Prevents R-loop formation by melting DNA–RNA hybrid with a 3′–5′ polarity |
DExH-Box Helicase 11 (DHX11) [78] | Converts RNA G-Quadruplex structures into R-Loops to promote IgH class switch recombination |
Werner Syndrome RecQ-Like Helicase (WRN) [64] | Protects the replication fork by preventing unscheduled R-loop formation |
DNA topology | |
Topoisomerase I/IIIB [55,67,79] | Involved in maintaining R-loop resolution by interacting with RNA-splicing and DNA-processing factors |
DNA repair and genome maintenance | |
Ataxia Telangiectasia Mutated (ATM)/Ataxia Telangiectasia And Rad3 Related (ATR) Kinase [57] | DNA-damage response (DDR) kinases that become activated when R-loop-mediated DNA damage occurs |
Breast Cancer Type 2 Susceptibility Protein (BRCA2/FANCD1) [41] | Binds to R-loops in response to dsDNA breaks to invite other DNA repair factors |
5. R-Loops Associated with Human Disease
- A.
- R-loops in Nucleotide Expansion Diseases
Diseases | Genes Associated with R-Loops |
---|---|
Aging [8,103,104] | SETX |
Alzheimer’s [8,103,104,105] | SETX, WW domain-containing oxidoreductase |
Aicardi–Goutières syndrome (AGS) [106] | TREX1, RNASEH2 |
AIDS-associated malignancies [107] | TREX complex |
Amyotrophic lateral sclerosis (ALS) [30,93,94] | C9orf72 and ATXN2 (GGGCCC)n, SETX |
Alternative lengthening of telomere (ALT)-dependent cancers [108] | TERRA complex |
Ataxia with oculomotor apraxia (AOA2) [7,109,110] | SETX |
Breast cancer [111,112,113,114] | BRAC1, BRAC2, Estrogen, SETX |
Burkitt’s lymphoma [84] | c-MYC, TRD3-TOP3B |
Colon cancer [115,116] | VIM |
Myotonic dystrophy type 1 (DM1) [100] | DMPK |
Embryonal tumors with multilayered rosettes (EMTR) [27] | C19MC |
Eosinophilic leukemia [15] | FIP1 |
Ewing’s sarcoma [117] | EWS-FLI, BRCA1 |
Frontotemporal dementia (FTD) [94] | C9orf7 (GGGCCC)n |
Fragile X syndrome type E (FRAXE) [101,102] | FRM2 (CCG)n |
Friedreich ataxia (FRDA) or fragile X syndrome type A (FRAXA) [96,97,98,99] | FXN (GAA)n, FRM1 (CCG)n |
Huntington’s disease (HD) [96,97,98,99] | HTT (CAG)n |
Infertility [118] | SETX |
Multiple myeloma [84,119] | c-MYC, TRD3-TOP3B, IFN |
Myelodysplastic syndromes [120] | U2AF1 (S34F), SRSF2 |
Polyglutamine-associated ataxias [95] | Multifactorial Nucleotide Expansion disorder |
Parkinson’s disease [8] | SETX |
Spinocerebellar ataxias (SCAs) [39] | ATXN1/2 (CAG)n |
Immunodeficiency, centromere instability, and facial anomalies (ICF) syndrome [121] | TERRA |
Wiskott–Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia [25] | XLT-WAS |
- B.
- R-loops in Neuronal Diseases
- C.
- R-loops in Cancer
- D.
- R-loops in other diseases
6. R-Loops as a Diagnostic Biomarker?
7. Emerging Technologies for Detecting R-Loops
8. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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R-Loop Binding Proteins | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
AQR | BUB3 | EWSR1 | RNASEH1 | RNASEH2A | RNASEH2C | THOC5 | TREX1 | DDX5 | EXOSC3 | |||
Transcription | YBX1 | |||||||||||
CTCF | ||||||||||||
LEO1 | ||||||||||||
XRN2 | ||||||||||||
SSU72 | ||||||||||||
POLR2B | ||||||||||||
POLR2C | ||||||||||||
Translation | CIRBP | |||||||||||
Cleavage and Polyadenylation | CPSF | CPSF1 | ||||||||||
CPSF2 | ||||||||||||
CPSF3 | ||||||||||||
CPSF4 | ||||||||||||
FIP1L1 | ||||||||||||
CSTF | CSTF1 | |||||||||||
CSTF2 | ||||||||||||
CSTF3 | ||||||||||||
CFIm | CPSF6 | |||||||||||
CPSF7 | ||||||||||||
NUDT21 | ||||||||||||
CFIIm | PCF11 | |||||||||||
CLP1 | ||||||||||||
Integrator complex | CPSF3L | |||||||||||
SYMPK | ||||||||||||
CSTF21 | ||||||||||||
WDR33 | ||||||||||||
RBBP6 | ||||||||||||
CPEB1 | ||||||||||||
PAP | PAPOLA | |||||||||||
PAPOLG | ||||||||||||
NC PAP | PAPD4 | |||||||||||
PAPD5 | ||||||||||||
PAPD7 | ||||||||||||
PABP | PABPC4 | |||||||||||
PABPN1 | ||||||||||||
PABPC1 | ||||||||||||
RNA processing | RBM5 | |||||||||||
PTBP1 | ||||||||||||
DDX39B | ||||||||||||
DDX23 | ||||||||||||
U2AF1 | ||||||||||||
SCAF1 | ||||||||||||
SRSF1 | ||||||||||||
SRSF3 | ||||||||||||
SRSF4 | ||||||||||||
RNPS1 | ||||||||||||
SCAF1 | ||||||||||||
XRN2 | ||||||||||||
DIS3L | ||||||||||||
SKIV2L2 | ||||||||||||
DCP2 | ||||||||||||
ZFP36 | ||||||||||||
KHSRP | ||||||||||||
TARBP2 | ||||||||||||
HNRNPH1 | ||||||||||||
Epigenetics | CHD1 | |||||||||||
PARN | ||||||||||||
Others | MAPK9 |
Detection Method | Method Name | Processing Method | Advantages | Disadvantages | |
---|---|---|---|---|---|
Wet lab techniques | |||||
Techniques for detecting R loop | S9.6 antibody staining DNA/RNA hybrid | Immunoprecipitation (IP)/Immunohistochemistry (IHC) [128]. | Immunostaining of DNA–RNA hybrid | Good signal, likely useful for the analysis of samples from tissue banks | Limited to the microscopic examination of R- loops |
DRIP [56,70,129,130,131] | Restriction digestion (RE) of genome followed by IP | Robust signal | Better resolution than IP/IHC but is still low | ||
DRIP-seq [31] | RE of genome followed by IP and dsDNA sequencing | Robust signal is widely adopted, and is easy to set up | Low resolution, no strand specificity, and cannot be used in situ | ||
S1-DRIP-seq [132] | Sonication of samples followed by IP and dsDNA sequencing | Higher resolution than DRIP-seq | No strand specificity and cannot be used in situ. S1 nuclease is delicate, and it is difficult to control the reaction, which may make it challenging to reproduce the data in clinical setting | ||
DRIPc-seq [133] | RE of genome followed by RNA sequencing | Strand-specific, high resolution | Not in situ, requires longer sample preparation, S9.6 may recognize dsRNA | ||
RDIP-seq [134] | Sonication of genome followed by RNA sequencing | Not in situ, tedious preparation | |||
Bis-DRIP-seq [135] | RE of genome followed by sequencing dsDNA with bisulfite conversions | Strand-specific, provides additional control to ensure S9.6 signal arises from an R-loop in situ | Requires many replicates and shows R-loop enrichment in promoter regions only | ||
qDRIP [136] | RE of genome followed by IP of DNA–RNA hybrid and synthetic DNA/RNA hybrid used as internal standards followed by dsDNA sequencing and quantification using internal standards as a reference. | Internal standards help with high-resolution, strand-specific sequencing | Spikes in hybrids shorter than 150 bp are unlikely to be useful for normalization. Additional spike-in may be required | ||
SMRF-seq [137] | RE of genome followed by IP of DNA/RNA hybrid and sequencing of dsDNA with bisulfite conversions at single molecule level | Strand-specific, single-molecule resolution, avoids biases inherent to read-count normalization by accurately profiling signals in regions unaffected by transcription inhibition thus providing accurate differential peak calling between conditions | As with any foot-printing method, SMF is agnostic to the distinguishing of DNA-binding proteincreating the footprints. | ||
Catalytically inactive RNase H | DRIVE-seq [31] | RE of genome followed by targeting catalytically inactive RNaseHs and dsDNA sequencing | Provides independent verification of some DRIP-seq results | Low enrichment, low resolution, reagent not commercially available, no strand specificity, not in situ | |
R-ChIP-seq [138] | Sonication followed by targeting catalytically inactive RNaseHs and ssDNA sequencing | Strand specific, in situ capture | Cell line must be engineered to express catalytically inactive RNase H construct, inactive RNase H may alter hybrid dynamics | ||
RNase H to guide micrococcal nuclease to R-loops | MapR [139] | Antibody-independent R-loop-profiling technique that utilizes RNase H to guide micrococcal nuclease to R-loops, which are subsequently cleaved, released, and identified by sequencing | Heavily based on CUT&RUN, a new and fast method to identify transcription factor binding sites genome-wide | Does not discriminate between the template and non-template strands and, therefore, cannot identify which DNA strand is involved in DNA–RNA hybrid formation. | |
Sensor that binds to R loop | R loop CUT&Tag [140] | Combines CUT&Tag and GST-His6-2×HBD (glutathione S-transferase–hexahistidine–2× hybrid-binding domain) tags as an artificial R loop hybrid sensor to specifically recognize the DNA–RNA hybrids. | Sensitive, reproducible and generates good resolution to sense the R-loop instead of capture strategies that largely contribute to disparities in the previous techniques including R-loop Mapping | Current form of R-loop CUT&Tag does not provide strand information about R loops | |
Techniques for detecting R loop and R loop-binding proteins | Fusion protein that binds to hybrid-binding domain (HBD) of RNaseH1 and an engineered variant of ascorbate peroxidase | RDProx (RNA–DNA Proximity Proteomics) [141] | Provides a snapshot of the R-loop-proximal proteome | In vivo labelling of R-loop-proximal proteins is performed, difficult to solubilize proteins that are amenable to the analysis can be identified, even transient spatiotemporal interactions with low affinity and transient interactions are detected. | Unable to distinguish between direct protein-binding or indirect proteins associated with RNA |
Staining of both DNA–RNA hybrid (S9.6 antibody)+ R-loop-binding proteins (Antibody specific to the desired protein) | IP/IHC (R-loops+ R-Loop-binding proteins) [124] | Immunostaining of DNA–RNA hybrid and their binding proteins | Fast analysis of pathology specimens | Low resolution and limited to routine microscopic analysis | |
Bioinformatics tools and databases | |||||
Techniques for detecting R loop | Structure-based detection and prediction based on existing wet-lab data | QmRLFS-finder [142] | Identifies three structural features of R loop including a short G-cluster-rich region (R-loop initiation zone or RIZ), a structurally non-specified linker (linker), and long downstream region that has high G-density R-loop elongation zone (or REZ) based on experimental data | User-friendly web server and stand-alone tool for rapid and accurate prediction of RLFSs in DNA or RNA sequences shows strong agreement with existing genes and genome-scale experimentally determined R-loops | Information is limited and an updated version needs to be integrated with the growing experimental data |
R loop tracker [143] | |||||
R-loop atlas [144] | About 63 million peaks called from 254 plant species by ssDRIP-seq and deepR-loopPre are available | User-friendly web server for plants species based on experimental data | Limited to plant species only | ||
Techniques for R loops and their binding proteins | R-loop DB [145] | Consists of computationally predicted R-loop-forming sequences (RLFSs) in human genic regions. Using the QmRLFS, the updated version of this database now has an increased number of RLFSs predicted in the human genes and in the genomes of other organisms | Provides comprehensive annotation of Ensembl RLFS-positive genes to study comparative evolution and genome-scale analyses, also R loop-binding proteins | Limited information and an updated version needs to be integrated with the growing experimental data |
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Khan, E.S.; Danckwardt, S. Pathophysiological Role and Diagnostic Potential of R-Loops in Cancer and Beyond. Genes 2022, 13, 2181. https://doi.org/10.3390/genes13122181
Khan ES, Danckwardt S. Pathophysiological Role and Diagnostic Potential of R-Loops in Cancer and Beyond. Genes. 2022; 13(12):2181. https://doi.org/10.3390/genes13122181
Chicago/Turabian StyleKhan, Essak S., and Sven Danckwardt. 2022. "Pathophysiological Role and Diagnostic Potential of R-Loops in Cancer and Beyond" Genes 13, no. 12: 2181. https://doi.org/10.3390/genes13122181
APA StyleKhan, E. S., & Danckwardt, S. (2022). Pathophysiological Role and Diagnostic Potential of R-Loops in Cancer and Beyond. Genes, 13(12), 2181. https://doi.org/10.3390/genes13122181