An R-loop is a three-stranded nucleic acid structure, composed of a DNA:

RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

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 Moiety may refer to: Chemistry * Moiety (chemistry), a part or functional group of a molecule ** Moiety conservation, conservation of a subgroup in a chemical species Anthropology * Moiety (kinship), either of two groups into which a society is ...

. 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 An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e. a region inside a gene."The notion of the cistron .e., gene ...

regions (which have been spliced out of the mRNA) form single-stranded DNA loops, as they cannot hybridize with complementary sequence in the mRNA.

History

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 Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respon ...

coding adenovirus

gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...

s contained DNA sequences that were not present in the mature mRNA. Roberts and Sharp were awarded the

Nobel Prize The Nobel Prizes ( ; sv, Nobelpriset ; no, Nobelprisen ) are five separate prizes that, according to Alfred Nobel's will of 1895, are awarded to "those who, during the preceding year, have conferred the greatest benefit to humankind." Alfre ...

in 1993 for independently discovering introns. After their discovery in adenovirus, introns were found in a number of

eukaryotic Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bact ...

genes such as the eukaryotic ovalbumin gene (first by the O'Malley laboratory, then confirmed by other groups), hexon DNA, and extrachromosomal

rRNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from riboso ...

genes of '' Tetrahymena thermophila''. In the mid-1980s, development of an

antibody An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and Viral disease, viruses. The antibody recognizes a unique m ...

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

R-loop mapping is a laboratory technique used to distinguish introns from

exon An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term ''exon'' refers to both the DNA sequence within a gene and to the corresponding sequenc ...

s in double-stranded DNA. These R-loops are visualized by

electron microscopy An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a ...

and reveal intron regions of DNA by creating unbound loops at these regions.

R-loops ''in vivo''

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 Endogenous substances and processes are those that originate from within a living system such as an organism, tissue, or cell. In contrast, exogenous substances and processes are those that originate from outside of an organism. For example, ...

R-loops, in conjunction with rapid advances in genetic

sequencing In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succi ...

technologies, inspired a blossoming of R-loop research in the early 2000's that continues to this day.

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. 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.

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

production. They also appear to play a role in protecting some active promoters from

methylation In the chemical sciences, methylation denotes the addition of a methyl group on a substrate, or the substitution of an atom (or group) by a methyl group. Methylation is a form of alkylation, with a methyl group replacing a hydrogen atom. These t ...

. The presence of R-loops can also inhibit transcription. Additionally, R-loop formation appears to be associated with “open”

chromatin Chromatin is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important ...

, characteristic of actively transcribed regions.

R-loops as genetic damage

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. R-loop accumulation has been associated with a number of diseases, including amyotrophic lateral sclerosis type 4 (ALS4), ataxia oculomotor apraxia type 2 (AOA2), Aicardi–Goutières syndrome, Angelman syndrome,

Prader–Willi syndrome Prader–Willi syndrome (PWS) is a genetic disorder caused by a loss of function of specific genes on chromosome 15. In newborns, symptoms include weak muscles, poor feeding, and slow development. Beginning in childhood, those affected become ...

, and cancer.

R-loops, Introns and DNA damage

Intron An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e. a region inside a gene."The notion of the cistron .e., gene ...

s are non-coding regions within

s 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 DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA da ...

. Introns reduce R-loop formation and DNA damage in highly expressed yeast genes. 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. Inserting an intron within an R-loop prone gene can also suppress R-loop formation and recombination. Bonnet et al. (2017) speculated that the function of introns in maintaining genetic stability may explain their evolutionary maintenance at certain locations, particularly in highly expressed genes.

References

{{reflist DNA RNA splicing