RNA biogenesis and RNA metabolism factors as R-loop suppressors: a hidden role in genome integrity

Abstract

Genome integrity relies on the accuracy of DNA metabolism, but as appreciated for more than four decades, transcription enhances mutation and recombination frequencies. More recent research provided evidence for a previously unforeseen link between RNA and DNA metabolism, which is often related to the accumulation of DNA-RNA hybrids and R-loops. In addition to physiological roles, R-loops interfere with DNA replication and repair, providing a molecular scenario for the origin of genome instability. Here, we review current knowledge on the multiple RNA factors that prevent or resolve R-loops and consequent transcription-replication conflicts and thus act as modulators of genome dynamics.

Keywords: DNA–RNA hybrids; R-loops; RNA biogenesis; RNA metabolism; genome instability; helicases.

Figure 1.

Types of DNA–RNA hybrids. (Top panel) R-loops are three-stranded structures containing DNA–RNA hybrids together with the displaced single-stranded DNA (ssDNA). The displaced ssDNA in R-loops can be targeted in vitro by bisulfite or in vivo by the activation-induced cytidine deaminase (AID). DNA–RNA hybrids can be detected with the anti-DNA–RNA hybrid antibody (S9.6), different inactive RNase H1 versions, or just its RNase hybrid-binding domain (RNase H*). DNA–RNA hybrids can also form at ssDNA gaps such as those transiently formed behind replication forks (middle panel) and at DNA double-strand breaks (DSBs) (bottom panel), which are referred to as break-induced DNA–RNA hybrids (BIRDHs).

Figure 2.

R-loops as a source of transcription-mediated genome instability. R-loops can be directly targeted by DNases, thus generating DNA breaks, albeit they mostly generate the stalling and subsequent breakage of replication forks. Transcription–replication conflicts (TRCs) can be either codirectional or head-on, with head-on having the strongest consequences in genome instability.

Figure 3.

A complex and multifactorial process for R-loop prevention. Different RNA biogenesis, processing, and export factors act as barriers for R-loop formation through their role as cotranscriptional RNA chaperons. Among them, the THO complex, together with other factors such as UAP56 and ALYREF, participates in the correct assembly of the mRNP particle, which is a protein-coated RNA molecule competent for export. Other RNA biogenesis factors, such as SRSF1 or the TREX-2 complex, and nucleoporins, such as TPR, also counteract R-loops by ensuring a proper RNA biogenesis and export or by inducing transcription termination, as in the case of SETX and XRN2. To ensure that only optimal mRNPs are exported to the cytoplasm, abortive and improperly processed transcripts are targeted by quality surveillance machineries that include the nuclear exosome and the TRAMP complex. Chromatin remodeling by SWI/SNF, FACT, or INO80 and supercoiling regulation by topoisomerases (Topo 1 and Topo 2) also contribute to counteract cotranscriptional R-loop accumulation. Importantly, functional cross-talk between the different processes, such as RNA biogenesis and chromatin modification, adds to R-loop suppression, as shown for the SIN3A histone deacetylase and the THO complex.

Figure 4.

R-loop and DNA–RNA hybrid resolvases. (Top) R-loops can be directly resolved by two different types of factors: DNA–RNA helicases that unwind DNA–RNA hybrids, and ribonucleases, which specifically cleave the RNA moiety. Only a selection of RNA helicases is shown, but a more extended list of DNA–RNA helicases is shown in Table 2. Contrary to RNase H1 and RNase H2, which can degrade the RNA within all DNA–RNA hybrids, the DICER ribonuclease only degrades the RNA of hybrids within R-loops. (Bottom) Some of these R-loop resolvases have also been shown to act at BIRDHs. Other factors that facilitate R-loop removal, such as DNA damage response (DDR) and chromatin remodeling and modification factors, are not shown. Rather than act directly on the R-loop, they likely promote repair of the R-loop-associated stalled replication fork or damaged DNA by their known DNA metabolic function, thus facilitating the release of the causative R-loop.