Regulatory R-loops as facilitators of gene expression and genome stability

Abstract

R-loops are three-stranded structures that harbour an RNA-DNA hybrid and frequently form during transcription. R-loop misregulation is associated with DNA damage, transcription elongation defects, hyper-recombination and genome instability. In contrast to such 'unscheduled' R-loops, evidence is mounting that cells harness the presence of RNA-DNA hybrids in scheduled, 'regulatory' R-loops to promote DNA transactions, including transcription termination and other steps of gene regulation, telomere stability and DNA repair. R-loops formed by cellular RNAs can regulate histone post-translational modification and may be recognized by dedicated reader proteins. The two-faced nature of R-loops implies that their formation, location and timely removal must be tightly regulated. In this Perspective, we discuss the cellular processes that regulatory R-loops modulate, the regulation of R-loops and the potential differences that may exist between regulatory R-loops and unscheduled R-loops.

Figure 1. Types of RNA–DNA hybrids.

RNA has the capacity to localize to genomic regions in a sequence specific manner and regulate downstream cellular processes (labelled in bold). R-loops are three-stranded structures harbouring an RNA–DNA hybrid and a displaced strand of DNA (highlighted in red). Non-R-loop forming RNA–DNA hybrids (highlighted in blue) are involved in different chromosomal transactions. At telomeres, the RNA moiety of the telomerase holoenzyme base-pairs with the 3´overhang at chromosome ends and provides a template for their extension. DNA replication, especially on the lagging strand, is dependent on the prior synthesis of small RNAs, which are used as polymerization primers by DNA polymerases. The cleavage of DNA-incorporated ribonucleoside monophosphates (rNMPs) on the 5´side serves as a signal for the DNA mismatch repair (MMR) machinery to distinguish the newly replicated strand from the template strand and ensure that the right mismatched base (here, ‘T’) is removed. R-loops are thought to form behind the RNA polymerase transcription machinery, where negative DNA supercoiling results in DNA unwinding, which provides an opportunity for the 5´end of the nascent transcript to base-pair with the template strand. Hybrid formation maybe be facilitated in the presence of G-rich sequences on the non-template strand, which may result in G4 structures. When RNA is produced from an exogenous source (such as a plasmid) or from a homologous chromosome or a distal homologous sequence, R-loops can also form in trans in a RAD51 recombinase-dependent manner. All RNA molecules are depicted in red, and DNA is depicted in blue. DSB, double strand break; G4, G-quadruplex secondary structure; NHEJ, non-homologous end joining; Pol II, RNA polymerase II.

Figure 2. R-loops across the genome.

R-loops have been mapped genome-wide in a number of species. The most prevalent predictor of R-loop presence is high transcriptional activity; indeed, R-loops are found at promoter regions, where they promote transcription by inducing DNA demethylation, and at transcription termination regions, where they promote transcription termination. Other features of R-loop-rich areas include high GC content and GC skew, g-quadruplex (G4) structures, antisense transcription and regions where the replication and transcription machineries collide. RNA–DNA hybrids (and perhaps R-loops) also form at sites of DNA damage, particularly at double-stranded breaks (DSBs), where they promote homologous recombination (HR)-mediated repair through the recruitment of breast cancer susceptibility protein 1 (BRCA1). ncRNA, non-coding RNAs; snoRNAs, small nucleolar RNAs; rDNA, ribosomal DNA.

Figure 3. R-loops as regulators of gene expression.

a. At the RASSF1A locus, the anti-sense long non-coding RNA (lncRNA) RASSF1 antisense RNA 1 (ANRASSF1) forms an R-loop in the promoter region, which can serve as a recruitment platform for Polycomb repressive complex 2 (PRC2) to silence RASSF1A expression through histone H3 Lys27 tri-methylation (H3K27me3). b. R-loops can be recognised by transcription factors to promote gene expression. At the VIM locus, the anti-sense lncRNA VIM antisense RNA 1 (VIM-AS1) promotes NF-kB recruitment through the formation of an R-loop at the transcription start site c. Histone modifiers such a the methyltransferase mixed-lineage leukaemia (MLL), are recruited to R-loops in the promoter region of the GATA3 gene to facilitate transcription through H3K4me3 deposition. d. At a subset of CpG island (CGI)-containing promoters, growth arrest and DNA damage protein 45A (GADD45) serves as an R-loop ‘reader’ that recruits ten-eleven translocation (TET) DNA demethylases, which demethylate the promoter DNA and activate transcription. Shown here is the case of the antisense lncRNA TCF21 antisense RNA inducing demethylation (TARID) and its cis-target gene transcription factor 21 (TCF21). The effect of demethylation may be enhanced by the exclusion of DNA (cytosine-5)-methyltransferases (DNMTs) such as DNMT3 from R-loop-rich CGIs. e. During differentiation of embryonic stem cells, the binding of chromatin modifiers with opposing functions (PRC1 and PRC2 versus TIP60–p400) can be dictated by R-loop occupancy, potentially resulting in histone modifications, such as histone H4 acetylation (H4ac) or incorporation of histone variants such as H2A.Z. f. Transcription of the TARID lncRNA, R-loop formation and transcription of the TCF21 mRNA proceed sequentially during the cell cycle. Tight temporal restriction of the formation of regulatory R-loops may be essential to prevent their detrimental effects.

Figure 4. R-loops promote transcription termination.

Shown are mechanisms by which R-loops may lead to pausing of elongating RNA polymerase II (Pol II) and transcription termination. a. Backtracking may arrest Pol II over the R-loop. Pol II oscillates between transcription elongation and backtracking. The presence of an R-loop may terminate the function of backtracked Pol II. b. During transcription, the R-loop remains fixed relative to the DNA helix, while elongating Pol II continues rotating along the helical path. The nascent transcript produced downstream of the R-loop must therefore wrap around the DNA, which may lead to Pol II arrest due to buildup of torsional stress. c. R-loops can promote the production of double-stranded RNA that recruits the RNase Dicer and the histone methyltransferase G9a, which forms a repressive chromatin environment that promotes Pol II pausing. Conversely, R-loop recognition and dissolving enzymes such as BRCA1, the RNA helicases senataxin (SETX), DHX9 and DDX5, and 5'-3' exoribonuclease 2 (XRN2) are also required for proper transcription termination at certain genes.

Figure 5. RNA–DNA hybrids can promote genome stability.

a. RNA–DNA hybrids at DNA double-stranded breaks (DSBs) may form from transcription stalling within a gene (not shown); from de novo transcription from a free 3´end at the break site, as in the case of damage induced long noncoding RNA (dilncRNA); or alternatively a pre-existing transcript may associate with the newly resected strand in trans. The hybrid attracts BRCA1 and eventually other repair factors (BRCA2, PALB2, XPG (not shown)). The hybrid is then removed by either senataxin, RNase H1, RNase H2 or DDX1 or DDX1 to allow RAD51 loading and homologous recombination (HR). b. At short telomeres, the accumulation of telomere repeat-containing RNA (TERRA) R-loops promotes repair through RAD51-mediated homology-dependent repair. It is uncertain whether TERRA can form R-loops in trans at short telomeres, and the R-loop removal pathway has not been identified c. The post-replicative formation of R-loops at centromeres leads to replication protein A (RPA)-dependent recruitment of the kinase ATR. ATR catalyses multiple phosphorylation events and stimulates Aurora B activity, which promotes accurate microtubule attachment to kinetochores and chromosome segregation. Pol II, RNA polymerase II.

Figure 6. Unscheduled vs. regulatory R-loops – a model.

a. We suggest that in normal conditions, potential sites of unscheduled-R-loop formation are free of RNA–DNA hybrids owing to either efficient hybrid prevention by RNA binding proteins, or to efficient R-loop removal. In the absence of R-loop prevention and removal, the dwell time of an R-loop increases, which can lead to replication stress, or to DNA damage on the, now vulnerable, displaced strand. Examples of proteins and protein complexes involved in R-loop removal or prevention are depicted. b. Regulatory R-loops may be associated with genomic features that allow R-loops to form more readily and with increased stability. Such features could include formation of stable G-quadruplexes (G4) on the non-template strand, GC skew, and a favourable chromatin environment. These features may also promote the formation of R-loops by trans-acting RNAs bound by RAD51, which promotes strand invasion at homologous sequences. Regulatory R-loops must also be resolved in order to prevent the very same problems that are associated with unscheduled R-loops. In the absence of R-loop removal factors, regulatory R-loops could be converted into unscheduled R-loops. DDX, DEAD-Box helicase; FACT, facilitates chromatin transcription; hnRNP, heterogeneous nuclear ribonucleoprotein; SETX, Senataxin; THO, suppressors of transcription defects of Hpr1 by overexpression, TOP1, Topoisomerase 1.