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R loops: new modulators of genome dynamics and function

Nature Reviews Genetics volume 16, pages 583–597 (2015)Cite this article

Subjects

Key Points

  • R loops consist of RNA–DNA hybrids that accumulate at preferred regions all over the genome, such as pericentromeric DNA, telomeres, ribosomal DNA or transcription termination regions.

  • R loops are an important source of replication stress and genome instability, which are hallmarks of cancer. As such, R loops seem to be a cause of tumorigenesis.

  • Cells have two ways to limit the number of R loops in the genome: mechanisms that remove R loops, such as ribonucleases and RNA–DNA helicases; and mechanisms that prevent R-loop accumulation, such as RNA-processing factors or topoisomerase 1.

  • R loops may play a part in transcription activation by directly affecting the chromatin structure of promoters and the recruitment of transcription or chromatin-remodelling factors. Such R loops may be formed by antisense non-coding RNAs.

  • R loops trigger chromatin condensation and heterochromatin formation, which may explain their ability to repress or silence gene expression and to stall replication-fork progression, putatively leading to replication-fork breakage as the main source of R-loop-mediated genome instability.

  • BRCA1 and BRCA2 have functions in double-strand break repair and/or replication-fork protection that may contribute to resolving intermediate structures such as stalled or broken replication forks that are generated as a consequence of R loops — thus these proteins facilitate the removal of R loops.

Abstract

R loops are nucleic acid structures composed of an RNA–DNA hybrid and a displaced single-stranded DNA. Recently, evidence has emerged that R loops occur more often in the genome and have greater physiological relevance, including roles in transcription and chromatin structure, than was previously predicted. Importantly, however, R loops are also a major threat to genome stability. For this reason, several DNA and RNA metabolism factors prevent R-loop formation in cells. Dysfunction of these factors causes R-loop accumulation, which leads to replication stress, genome instability, chromatin alterations or gene silencing, phenomena that are frequently associated with cancer and a number of genetic diseases. We review the current knowledge of the mechanisms controlling R loops and their putative relationship with disease.

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Figure 1: Cellular activities limiting R-loop accumulation.
Figure 2: R loops as regulators of transcription.
Figure 3: A model to explain replication-mediated R-loop-induced genome instability.

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Acknowledgements

The authors thank T. García-Muse for critical reading of the manuscript and D. Haun for style supervision. Research in A.A.'s laboratory is funded by grants from the Spanish Ministry of Economy and Competitiveness, Junta de Andalucía, European Union (FEDER), Worldwide Cancer Research and PharmaMar. The authors apologize to those whose work could not be cited owing to space limitations.

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  1. Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Av. Américo Vespucio s/n, Seville, 41092, Spain

    José M. Santos-Pereira & Andrés Aguilera

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  1. José M. Santos-Pereira
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Correspondence to Andrés Aguilera.

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PowerPoint slides

Glossary

G-quartets

Planar structures formed by four intra-strand Gs paired with each other.

Genome instability

A cellular pathological condition by which cells undergo mutations, recombination, chromosome rearrangements or chromosome loss at frequencies substantially higher than wild-type levels.

Holliday junctions

Double-stranded, cruciform structures formed as intermediates of recombinational repair in which both recombining DNA molecules are covalently linked.

Recombinational repair

A double-strand break repair that is active during the S–G2 phases of the cell cycle and that uses information from a homologous sequence, normally the sister chromatid, to copy DNA.

Okazaki fragment

A discrete fragment created by synthesis of the DNA lagging strand during replication. Okazaki fragments are primed by a short RNA in the form of an RNA–DNA hybrid.

Negative supercoiling

Under-winding of a DNA strand, typically occurring behind an elongating RNA polymerase.

Topoisomerase

(TOP). An enzyme family that can remove (or create) supercoiling in duplex DNA by making transitory breaks in one strand (type 1 TOPs) or both strands (type 2 TOPs) of the DNA backbone.

D loops

(Displacement loops). DNA structures consisting of a main double-stranded DNA that has been separated by a third DNA strand complementary to one of the main strands and that by pairing with it displaces the other main strand.

Bisulfite mutagenesis

A method to mutagenize DNA with sodium bisulfite, which acts exclusively over single-stranded DNA and, therefore, serves to determine whether a particular DNA sequence is present in vivo in a single-stranded form (as is the case for the strand displaced by the RNA in the R loop) or in a double-stranded form.

DNA damage response

(DDR). A general cellular response consisting of DNA damage sensing, activation of different checkpoints, the action of the appropriate DNA repair pathway and the arrest of the cell cycle.

Multicopy suppressors

Genes that are able to suppress the phenotype conferred by specific mutations when they are present in high-copy-number plasmids.

CpG islands

CpG-rich regions that are usually unmethylated and localized at the 5′ end of genes, where they function as promoter elements.

GC skew

Asymmetry in the distribution of Gs and Cs between DNA strands, with an overrepresentation of Gs in the non-transcribed DNA strand.

Heterochromatin

A chromosomal region with highly compacted chromatin that is more refractory to the action of enzymes, has a general repressive action on gene transcription and replicates late in the cell cycle.

Homeodomain

A DNA-binding domain that is characteristic of homeobox proteins involved in transcription regulation. It consists of a 60-amino-acid helix–turn–helix structure with three α-helices connected by loop regions.

RNA interference

(RNAi). A mechanism of gene silencing that relies on short non-coding RNAs that have the ability to repress chromatin with the help of additional ancillary factors.

Replication stress

Any condition in which replication progression slows down and/or stalls, commonly leading to genome instability.

Break-induced replication

(BIR). A mechanism of recombinational repair in which a one-ended double-strand break invades a homologous DNA sequence that is used as template for DNA synthesis to complete repair.

DNA combing

A technique used to produce stretched DNA fibres for multiple applications, including the study of DNA replication by immune detection of modified nucleotides.

Common fragile site

A specific chromosome region that has gaps or constrictions that are visible under the microscope and that tend to break on exposure to replication stress.

Backtracked RNA polymerase

An intermediate state in which an arrested RNA polymerase moves back to allow cleavage of the last ribonucleotide incorporated into the nascent RNA, thus allowing transcription resumption.

Nucleotide excision repair

(NER). A conserved DNA repair pathway that recognizes adducts and repairs them by excision of a short oligonucleotide containing the damage.

Fanconi anaemia pathway

A DNA repair pathway that works on replication forks stalled at interstrand crosslinks and other lesions that block replication.

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Santos-Pereira, J., Aguilera, A. R loops: new modulators of genome dynamics and function. Nat Rev Genet 16, 583–597 (2015). https://doi.org/10.1038/nrg3961

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