Out of balance: R-loops in human disease

Abstract

R-loops are cellular structures composed of an RNA/DNA hybrid, which is formed when the RNA hybridises to a complementary DNA strand and a displaced single-stranded DNA. R-loops have been detected in various organisms from bacteria to mammals and play crucial roles in regulating gene expression, DNA and histone modifications, immunoglobulin class switch recombination, DNA replication, and genome stability. Recent evidence suggests that R-loops are also involved in molecular mechanisms of neurological diseases and cancer. In addition, mutations in factors implicated in R-loop biology, such as RNase H and SETX (senataxin), lead to devastating human neurodegenerative disorders, highlighting the importance of correctly regulating the level of R-loops in human cells. In this review we summarise current advances in this field, with a particular focus on diseases associated with dysregulation of R-loop structures. We also discuss potential therapeutic approaches for such diseases and highlight future research directions.

Figure 1. History of R-loop research.

The diagram depicts major developments in the R-loop field and diseases associated with R-loop dysregulation.

Figure 2. R-loops and human diseases.

The diagram depicts the role of R-loops in human diseases. Loss of wild type protein function is depicted by red crosses. A. Ataxia and motor neuron diseases. Mutations in human RNA/DNA helicase senataxin are associated with AOA2/ALS4 disorders and lead to R-loop accumulation and defects in transcriptional termination by Pol II , the maintenance of genome integrity , meiotic recombination during spermatogenesis, gene silencing during meiotic sex chromosome inactivation , and neuronal differentiation . B. Aicardi-Goutières syndrome (AGS). AGS is associated with mutations in all three subunits of RNase H2, ssDNA 3′–5′ exonuclease TREX1 (DNASEIII), dsRNA-editing enzyme ADAR1, and dNTP triphosphatase SAMHD1; these trigger accumulation of unprocessed nucleic acids, including genomic DNA with incorporated ribonucleotides, R-loops, and retroelement-derived nucleic acids, and result in the immune response characteristic of AGS . C. Trinucleotide expansion diseases. R-loops form over expanded repeats and result in decreased initiation and elongation of RNA Pol II and formation of repressive chromatin marks, which silence the host gene containing expanded repeats . D. Genome instability in cancer. Loss of proteins protecting against abnormal R-loop accumulation, such as FIP1L1, leads to genome instability, one hallmark of cancer . Yellow stars denote double-stranded DNA breaks. E. AID-mediated mutagenesis and translocations in cancer. Single-stranded DNA in R-loops is a substrate for cytidine deamination by activation-induced cytidine deaminase, leading to mutagenesis as indicated by orange stars , . These mutations can cause DSB formation, leading to chromosomal translocations. The IgH/c-MYC translocation brings the strong IgH enhancers, shown as yellow box, close to c-MYC, leading to its overexpression in Burkitt's lymphoma . Transcription of IgH/c-MYC starts from a previously inactive promoter downstream of the translocation break point. The IgH locus is depicted in blue, c-MYC gene is in grey. The translocation breakpoint is indicated by a dashed black line. F. Senescence. R-loops formed by the noncoding RNA TERRA accumulate at telomeres in cells deficient of Hpr1 and RNase H. In the absence of telomerase, these R-loops promote Rad52-dependent telomere elongation and delayed senescence. In the absence of telomerase and Rad52, R-loops promote telomere shortening and premature senescence .

Figure 3. Potential R-loop-based therapeutic approach in Angelman Syndrome (AS).

A. Neuronal expression of the paternal ncRNA Ube3a-ATS represses paternal Ube3a gene in cis . DNA methylation of the Snord116 locus on the maternal allele prevents Ube3a-ATS transcription, resulting in Ube3a expression from the maternal allele. Transcriptional repression is indicated by red crosses. B. R-loop-mediated re-activation of silent paternal Ube3a gene provides a targeted therapy for AS. Deletion leading to the loss of maternal Ube3a expression detected in AS is indicated by the red dashed line. Topotecan treatment increases R-loop levels over the Snord116 locus, resulting in chromatin decondensation, inhibition of Pol II transcription through Ube3a-ATS, and increased expression of Ube3a from the paternal allele .