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 (ssDNA). 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.

History

An illustration showing how a DNA-mRNA hybrid forms R-Loops in the regions where introns have been removed through splicing exons.

R-looping was first described in 1976.^[1] 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.^[2][3] 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) ^[4][5] hexon DNA,^[6] and extrachromosomal rRNA 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] Additionally, R-loop formation appears to be associated with “open” chromatin, characteristic of actively transcribed regions.^[18][19]

R-loops as genetic damage

When unscheduled R-loops form, they can cause damage by a number of different mechanisms. Exposed ssDNA 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 (DSBs).^[20] As well, R-loops may induce unscheduled replication by acting as a primer.^[21][22]

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, and cancer.^[23]

See also

• DRIP-seq
• Ribonuclease H
• Immunoglobulin class switching
• DNA replication

References

1. Thomas M, White RL, Davis RW. “Hybridization of RNA to double-stranded DNA: formation of R-loops”. Proc. Natl Acad. Sci. USA. 1976; 73:2294–2298.
2. Berget SM, Moore C, Sharp PA. “Spliced segments at the 5′ terminus of adenovirus 2 late mRNA”. Proc Natl Acad Sci USA. 1977; 8:3171–3175. doi: 10.1073/pnas.74.8.3171.
3. Chow LT, Gelinas RE, Broker TR, Roberts RJ. “An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA”. Cell. 1977; 12(1):1-8.
4. Lai EC, Woo SL, Dugaiczyk A, Catterall JF, O'Malley BW (1978) "The ovalbumin gene: structural sequences in native chicken DNA are not contiguous" Proc Natl Acad Sci U S A. 1978 May;75(5):2205-9
5. O’Hare K, Breathnach R, Benoist C, Chambon P (1979). “No more than seven interruptions in the ovalbumin gene: comparison of genomic and double-stranded cDNA sequences.” Oxford Journals, 7(2): 321-334.
6. Berget SM, Moore C, Sharp PA. “Spliced segments at the 5′ terminus of adenovirus 2 late mRNA”. Proc Natl Acad Sci USA. 1977; 8:3171–3175. doi: 10.1073/pnas.74.8.3171.
7. Cech TR and Rio DC (1979). “Localization of transcribed regions on extrachromosomal ribosomal RNA genes of Tetrahymena thermophila by R-loop mapping.” Proc Natl Acad Sci USA. 76, (10): 5051-5055.
8. Boguslawski SJ et al (1986). "Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids". J Immunol Methods. 1;89(1):123-30.
9. Woolford, John L., Jr.; Rosbash, Michael (1979). (The use of R-looping for structural gene identification and mRNA purification (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC327867/pdf/nar00448-0111.pdf)”. Nucleic Acids Research. 6 (7):2483-97. PMC327867
10. King RC, Stansfield WD, Mulligan PK (2007). A Dictionary of Genetics. Oxford University Press 7.
11. Itoh T, Tomizawa J (1980). “Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H.” Proc Natl Acad Sci USA 77:2450–2454.
12. Drolet M, Bi X, Liu LF (1994). “Hypernegative supercoiling of the DNA template during transcription elongation in vitro.” J Biol Chem 269: 2068–2074.
13. Groh M, Gromak N (2014) Out of Balance: R-loops in Human Disease. PLoS Genet 10(9): e1004630. doi:10.1371/journal.pgen.1004630
14. Cerritelli SM, Crouch RJ (2009). "Ribonuclease H: the enzymes in eukaryotes." FEBS J. 279:1494-1505.
15. Chan YA, Aristizabal MJ, Lu PY, Luo Z, Hamza A, Kobor MS, Stirling PC, Hieter P: Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip. PLOS Genet 2014, 10:e1004288.
16. Roy D, Yu K, Lieber MR: Mechanism of R-loop formation at immunoglobulin class switch sequences. Mol Cell Biol 2008, 28:50-60.
17. Ginno PA, Lott PL, Christensen HC, Korf I, Chedin F: R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol Cell 2012, 45:814-825.
18. Castellano-Pozo M, Santos-Pereira JM, Rondon AG, Barroso S, Andujar E, Perez-Alegre M, Garcia-Muse T, Aguilera A: R loops are linked to histone H3 S10 phosphorylation and chromatin condensation. Mol Cell 2013, 52:583-590.
19. Costantino L, Koshland D (2015). “The Yin and Yang of R-loop biology.” Curr Opin Cell Biol. 34:39-45. doi: 10.1016/j.ceb.2015.04.008.
20. Sollier J, Cimprich KA (2015). "Breaking bad: R-loops and genome integrity." Trends Cell Biol. S0962-8924(15)00093-8. doi: 10.1016/j.tcb.2015.05.003.
21. Itoh T, Tomizawa J (1980). “Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H.” Proc Natl Acad Sci USA 77:2450–2454.
22. Costantino L, Koshland D (2015). “The Yin and Yang of R-loop biology.” Curr Opin Cell Biol. 34:39-45. doi: 10.1016/j.ceb.2015.04.008.
23. Groh M, Gromak N (2014) Out of Balance: R-loops in Human Disease. PLoS Genet 10(9): e1004630. doi:10.1371/journal.pgen.1004630