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 DNA loops, as they cannot hybridize with complementary sequence in the mRNA.[1]
History
R-looping was first described in 1976.[2] Independent R-looping studies from the laboratories of
Richard J. Roberts and
Phillip A. Sharp showed that
protein coding
adenovirusgenes contained DNA sequences that were not present in the mature mRNA.[3][4] 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),[5][6]hexon DNA,[3] and
extrachromosomalrRNA genes of Tetrahymena thermophila.[7]
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.[8]
R-loop mapping
R-loop mapping is a laboratory technique used to distinguish introns from
exons in double-stranded DNA.[9] These R-loops are visualized by
electron microscopy and reveal intron regions of DNA by creating unbound loops at these regions.[10]
R-loops in vivo
The potential for R-loops to serve as replication primers was demonstrated in 1980.[11] In 1994, R-loops were demonstrated to be present in vivo through analysis of plasmids isolated from E. coli mutants carrying mutations in
topoisomerase.[12] 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.[13]
Regulation of R-loop formation and resolution
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.[14] 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.[15]
Roles of R-loops in genetic regulation
R-loop formation is a key step in
immunoglobulin class switching, a process that allows activated
B cells to modulate
antibody production.[16] They also appear to play a role in protecting some active
promoters from
methylation.[17] The presence of R-loops can also inhibit transcription.[18] Additionally, R-loop formation appears to be associated with “open”
chromatin, characteristic of actively transcribed regions.[19][20]
R-loops as genetic damage
When unscheduled R-loops form, they can cause damage by a number of different mechanisms.[21] 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.[22] As well, R-loops may induce unscheduled replication by acting as a
primer.[11][20]
Introns are non-coding regions within
genes that are transcribed along with the coding regions of genes, but are subsequently removed from the
primary RNA transcript by
splicing. Actively transcribed regions of
DNA often form R-loops that are vulnerable to
DNA damage. Introns reduce R-loop formation and DNA damage in highly expressed yeast genes.[23] Genome-wide analysis showed that intron-containing genes display decreased R-loop levels and decreased DNA damage compared to intron-less genes of similar expression in both yeast and humans.[23] Inserting an intron within an R-loop prone gene can also suppress R-loop formation and
recombination. Bonnet et al. (2017)[23] speculated that the function of introns in maintaining genetic stability may explain their evolutionary maintenance at certain locations, particularly in highly expressed genes.
^Boguslawski SJ, Smith DE, Michalak MA, Mickelson KE, Yehle CO, Patterson WL, Carrico RJ (May 1986). "Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids". Journal of Immunological Methods. 89 (1): 123–30.
doi:
10.1016/0022-1759(86)90040-2.
PMID2422282.