R-Loops as Cellular Regulators and Genomic Threats

Abstract

During transcription, the nascent RNA strand can base pair with its template DNA, displacing the non-template strand as ssDNA and forming a structure called an R-loop. R-loops are common across many domains of life and cause DNA damage in certain contexts. In this review, we summarize recent results implicating R-loops as important regulators of cellular processes such as transcription termination, gene regulation, and DNA repair. We also highlight recent work suggesting that R-loops can be problematic to cells as blocks to efficient transcription and replication that trigger the DNA damage response. Finally, we discuss how R-loops may contribute to cancer, neurodegeneration, and inflammatory diseases and compare the available next-generation sequencing-based approaches to map R-loops genome wide.

Figure 1.

R-loop resolution and suppression mechanisms. R-loops form co-transcriptionally when nascent RNA (red) hybridizes with DNA (black), generating an RNA-DNA hybrid and displaced ssDNA. R-loops are suppressed by processing/splicing factors which coat nascent RNA, elongation factors that ensure processive transcription, or transcriptional repression at certain repeat sequences. Once formed, a variety of helicases (blue) and other factors such as the FANCM translocase, RNase H family nucleases (RNH), or the replisome (not shown) may remove the hybrid, restoring processive transcription. Not all factors implicated in this process have been illustrated for clarity, and many factors have not been examined in biochemical assays to support the proposed activity. Factors outlined in dotted lines show R-loop resolution in cellular assays, but have not been shown to biochemically resolve RNADNA hybrids in vitro.

Figure 2.

R-loops participate in gene regulation. A) At promoters, R-loops activate transcription by preventing binding of transcriptional repressors or DNA methylating enzymes (DNMT), or by acting as binding sites for transcription factors (top left). Alternatively, R-loops repress transcription by blocking transcription factor binding (bottom left). B) At terminators, R-loops facilitate efficient transcription termination by promoting RNAP II pausing and cleavage of the transcript from its template either by recruiting R-loop resolution helicases and RNases (bottom right), or by recruiting the RNAi silencing machinery (top right).

Figure 3.

R-loops at transcription-replication collisions. Collisions of a replication fork with an R-loop can resolve the R-loop when replication forks are co-directional with transcription, or stabilize them when replication forks collide with the R-loop head-on. The MCM replicative helicase complex may directly unwind RNA-DNA hybrids in the co-directional orientation.

Figure 4.

Transcription stress and R-loops. A) Transcription stress may arise from a stalled RNA polymerase associated with an R-loop (left) or upstream polymerases that are stalled from collisions with the R-loop (right). B) Two ways that NER processing may convert an R-loop to a DSB: either NER enzymes XPG and XPF cut the hybridized DNA, leading to a single-strand gap that is processed into a DSB by replication (top), or NER enzymes cut non-canonically to directly create a DSB at the 3’ or 5’ end of the R-loop (bottom).

Figure 5.

R-loops drive DNA damage responses. A) R-loops may cause DSBs and canonical ATM activation through three mechanisms: damage to the displaced ssDNA that is converted to a DSB by DNA replication, nucleolytic processing of the R-loop or fork stalled by R-loops, or collision of the replication fork with a backtracked RNA polymerase. B) R-loops may activate ATR in head-on collisions by stalling replication forks, which then accumulate RPA and signal to ATR through the canonical pathway. C) Non-canonical DNA damage response pathway activation could occur when polymerases stalled at R-loops activate ATM, or when RPA accumulates on the displaced ssDNA and activates ATR.

Figure 6.

R-loops participate in DNA repair at breaks. A) DNA breaks, either ssDNA nicks (left) or DSBs (right), create free 3’-DNA ends, promoting the annealing of RNA to DNA to form hybrids. B) In yeast, R-loops form at DSBs and initiate repair by HR. Subsequent R-loop resolution by Sen1 or RNase H regulates the extent of DNA end resection and subsequent binding of RPA (left). In the absence of Sen1 or RNase H (right), RNA-DNA hybrids persist, blocking DNA resection and RPA binding and leading to chromosomal rearrangements. C) In human cells RNA-DNA hybrids formed at a subset of DSBs recruit Rad52, which further recruits XPG and BRCA1. XPG-mediated R-loop processing initiates DNA resection and repair by HR, and suppresses aberrant NHEJ. D) SETX is recruited to DSBs and resolves RNA-DNA hybrids at active genes. SETX regulates γH2AX spreading and Rad51 foci formation promoting DSB repair and suppressing chromosomal translocations. E) Rad52 promotes the formation of RNA- DNA hybrids that facilitate RNA-templated HR by bridging two DNA ends. The DNA ends are ligated, and the RNA strand is removed by RNase H.