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Review
. 2014 Jul 1;28(13):1384-96.
doi: 10.1101/gad.242990.114.

A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression

Konstantina Skourti-Stathaki  1 , Nicholas J Proudfoot  1
Affiliations
Review

A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression

Konstantina Skourti-Stathaki et al. Genes Dev. .

Abstract

R loops are three-stranded nucleic acid structures that comprise nascent RNA hybridized with the DNA template, leaving the nontemplate DNA single-stranded. R loops form naturally during transcription even though their persistent formation can be a risky outcome with deleterious effects on genome integrity. On the other hand, over the last few years, an increasingly strong case has been built for R loops as potential regulators of gene expression. Therefore, understanding their function and regulation under these opposite situations is essential to fully characterize the mechanisms that control genome integrity and gene expression. Here we review recent findings about these interesting structures that highlight their opposite roles in cellular fitness.

Keywords: R loops; gene expression; genome integrity.

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Figures

Figure 1.
Figure 1.
R loops as a source of DNA damage. Nascent transcripts behind elongating Pol II can invade the DNA duplex and hybridize with the DNA template strand. The RNA/DNA hybrid so formed displaces the nontemplate strand, and this three-stranded structure constitutes an R loop. R loops can cause genomic instability in different ways. First, the displaced ssDNA can act as a substrate to DNA-damaging agents, deaminases (AID), and repair enzymes (APE and BER), leading to DNA lesions and nicks. Second, G4 structures forming on the G-rich nontemplate strand can generate susceptible sites for nucleases. Finally, transcription elongation machinery impeded by stable R loops can cause replication–transcription collisions, leading to DNA recombination and DSBs. Points of contact between the DNA strand and nascent RNA indicate R-loop formation, whereas points of contact within the ssDNA indicate G4 structures. Pol II is shown as a blue icon, with an arrow indicating transcription direction. Nucleosomes are shown in green. The diagram is not drawn to scale.
Figure 2.
Figure 2.
Diverse protection mechanisms against R-loop formation. Two types of surveillance factors have been identified: factors that prevent formation of R loops and factors that actively remove them. DNA topoisomerase enzymes suppress R-loop formation by relaxing the negative supercoiling behind elongating Pol II. The THO complex (blue circle) facilitates efficient packaging of nascent RNA into messenger ribonucleotide proteins (mRNPs), preventing R-loop formation. Splicing and 3′ end processing factors associate with nascent RNA and prevent R loops. RNA/DNA helicases and RNase H enzymes remove R loops once formed. DNA is shown as gray and black lines, and RNA is shown as a red line. Dotted lines indicate the site of action of different factors. The diagram is not drawn to scale.
Figure 3.
Figure 3.
Rad51 can promote cis and trans R loops. The HR factor Rad51 can promote strand exchange, ultimately leading to cotranscriptional R-loop formation (cis R-loop). Trans R loops can also be mediated by Rad51. As shown in the diagram, trans RNA may target the ssDNA as part of a pre-existing R loop. Alternatively, trans RNA could target dsDNA if local unwinding of the DNA duplex occurs by mechanisms such as DNA replication. Such trans R loops are associated with the popular CRISPR–Cas9 system in which CRISPR guide RNA hybridizes with target DNA loci generating targeted DNA breaks. Trans R loops can also occur between ncRNAs and homologous DNA, ultimately leading to transcriptional gene silencing. DNA is shown as gray and black lines, and RNA is shown as a red line. The diagram is not drawn to scale.
Figure 4.
Figure 4.
R loops are enriched at both gene ends. In human protein-coding genes, R loops form over unmethylated CpG island promoters with positive GC skew and G-rich termination regions. Promoter-enriched R loops could activate gene expression, whereas terminator-enriched R loops promote transcriptional termination by facilitating Pol II pausing downstream from the poly(A) signal. Transcription start site (TSS), transcription termination site (TTS), and poly(A) (pA) signal are shown. Colored shading indicates peaks of R loops over 5′ and 3′ gene ends. The diagram is not drawn to scale.
Figure 5.
Figure 5.
R loops transcriptionally regulate ncRNAs. (A) In plants, COOLAIR antisense lncRNA controls the expression of the FLC gene. R loops form over the promoter region of COOLAIR and are stabilized by the ssDNA-binding protein AtNDX. This causes transcriptional repression of COOLAIR and, ultimately, activation of the FLC gene. (B) In human neuronal cells, topoisomerase inhibitor topotecan causes accumulation of R loops in the G-rich termination region of the Snord116 gene. This causes chromatin decondensation and blocks read-through transcription that otherwise forms the Ube3a antisense transcript. This activates the expression of the Ube3a sense transcript. Arrows indicate the direction of transcription. For simplicity, nucleosomes are omitted. The diagram is not drawn to scale.
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