R-loop

An R-loop is a three-stranded nucleic acid structure, composed of a DNA:RNA hybrid and the associated non-template single-stranded DNA. R-loops may be formed in a variety of circumstances, and may be tolerated or cleared by cellular components. The term 'R-loop' was given to reflect the similarity of these structures to D-loops; the 'R' in this case represents the involvement of an RNA moiety. An R-loop is a three-stranded nucleic acid structure, composed of a DNA:RNA hybrid and the associated non-template single-stranded DNA. R-loops may be formed in a variety of circumstances, and may be tolerated or cleared by cellular components. The term 'R-loop' was given to reflect the similarity of these structures to D-loops; the 'R' in this case represents the involvement of an RNA moiety. In the laboratory, R-loops may also be created by the hybridization of mature mRNA with double-stranded DNA under conditions favoring the formation of a DNA-RNA hybrid; in this case, the intron regions (which have been spliced out of the mRNA) form single-stranded loops, as they cannot hybridize with complementary sequence in the mRNA. R-looping was first described in 1976. Independent R-looping studies from the laboratories of Richard J. Roberts and Phillip A. Sharp showed that protein coding adenovirus genes contained DNA sequences that were not present in the mature mRNA. Roberts and Sharp were awarded the Nobel Prize in 1993 for independently discovering introns. After their discovery in adenovirus, introns were found in a number of eukaryotic genes such as the eukaryotic ovalbumin gene (first by the O'Malley laboratory, then confirmed by other groups), hexon DNA, and extrachromosomal rRNA genes of Tetrahymena thermophila. In the mid-1980s, development of an antibody that binds specifically to the R-loop structure opened the door for immunofluorescence studies, as well as genome-wide characterization of R-loop formation by DRIP-seq. R-loop mapping is a laboratory technique used to distinguish introns from exons in double-stranded DNA. These R-loops are visualized by electron microscopy and reveal intron regions of DNA by creating unbound loops at these regions. The potential for R-loops to serve as replication primers was demonstrated in 1980. In 1994, R-loops were demonstrated to be present in vivo through analysis of plasmids isolated from E. coli mutants carrying mutations in topoisomerase. This discovery of endogenous R-loops, in conjunction with rapid advances in genetic sequencing technologies, inspired a blossoming of R-loop research in the early 2000s that continues to this day. RNaseH enzymes are the primary proteins responsible for the dissolution of R-loops, acting to degrade the RNA moiety in order to allow the two complementary DNA strands to anneal. Research over the past decade has identified more than 50 proteins that appear to influence R-loop accumulation, and while many of them are believed to contribute by sequestering or processing newly transcribed RNA to prevent re-annealing to the template, mechanisms of R-loop interaction for many of these proteins remain to be determined. R-loop formation is a key step in immunoglobulin class switching, a process that allows activated B cells to modulate antibody production. They also appear to play a role in protecting some active promoters from methylation. The presence of R-loops can also inhibit transcription. Additionally, R-loop formation appears to be associated with “open” chromatin, characteristic of actively transcribed regions. When unscheduled R-loops form, they can cause damage by a number of different mechanisms. Exposed single-stranded DNA can come under attack by endogenous mutagens, including DNA-modifying enzymes such as activation-induced cytidine deaminase, and can block replication forks to induce fork collapse and subsequent double-strand breaks. As well, R-loops may induce unscheduled replication by acting as a primer.