R loop

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An R-Loop is a three-stranded nucleic acid structure that consists of a DNA: RNA hybrid and the associated single-stranded, untranslated DNA . R-Loops can be formed under various circumstances and tolerated or released by cellular components. The term "R-loop" has been used to reflect the similarity of these structures to D-loops ; the "R" in this case represents the involvement of an RNA unit.

In the laboratory, R-loops can also be generated by hybridizing mature mRNA with double-stranded DNA under conditions that favor 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 because they cannot hybridize with complementary sequence in the mRNA.

history

The illustration shows how a DNA-mRNA hybrid forms R-loops in the areas where introns have been removed by splicing exons.

The R-loop mechanism was first described in 1976. Independent studies of R-loops from the labs of Richard J. Roberts and Phillip A. Sharp showed that a protein that encoded adenovirus genes contained DNA sequences that were not present in the mature mRNA. Roberts and Sharp received the 1993 Nobel Prize for the independent discovery of introns. After their discovery in adenovirus, introns have been found in a number of eukaryotic genes, such as the eukaryotic ovalbumin gene (confirmed first by the O'Malley laboratory, then by other groups), hexon DNA, and extrachromosomal rRNA genes from Tetrahymena thermophila .

In the mid-1980s, the development of an antibody that specifically binds to the R-loop structure opened the door to immunofluorescence studies and the genome-wide characterization of R-loop formation using DRIP-Seq .

R-loop mapping

R-loop mapping is a laboratory technique used to distinguish introns and exons in double-stranded DNA. These R-loops are visualized using an electron microscope and show intron areas of the DNA by creating unbound loops at these areas.

R-loops in vivo

The potential of R-loops as a primer for replication was demonstrated in 1980. In 1994 it was proven that R-loops are present in vivo. Here, plasmids were analyzed that were isolated from E. coli mutants that carry mutations in the topoisomerase. This discovery of endogenous R-loops, coupled with the rapid advances in genetic sequencing technology, led to a heyday of R-loop research in the early 2000s that continues to this day.

Regulation of R-loop formation and resolution

RNase-H enzymes are the most important proteins responsible for breaking up R-loops and breaking down the RNA part in order to allow the two complementary DNA strands to hybridize. Research over the past decade has identified more than 50 proteins that appear to affect R-loop accumulation. While many of them are believed to help by sequestering or processing newly transcribed RNA to prevent re-hybridization to the DNA template, the mechanisms of R-loop interaction for many of these proteins have yet to be determined.

Role of R-Loops in Genetic Regulation

The formation of R-loops is an important step in switching immunoglobulin class , a process that enables 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. In addition, the R-loops appear to be associated with the "open" chromatin that is characteristic of actively transcribed regions.

Genetic damage from R-loops

When R-Loops form unplanned, they can do damage through a number of different mechanisms. Exposed single-stranded DNA can be attacked by endogenous mutagens, including DNA-modifying enzymes such as activation-induced cytidine deaminase. It can also block replication forks to induce their collapse and subsequent double-strand breaks. In addition, R-loops can induce unplanned replication by acting as primers.

The accumulation of R-loops has been linked to a number of diseases including amyotrophic lateral sclerosis type 4 (ALS4), oculomotor apraxia type 2 (AOA2), Aicardi-Goutières syndrome , Angelman syndrome , Prader-Willi syndrome, and cancer .

R-loops, introns and DNA damage

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 areas of DNA often form R-loops, which are prone to DNA damage. Introns reduce the formation of R-loops and DNA damage in highly expressed yeast genes. A genome-wide analysis showed that intron-containing genes, compared to intron-free genes with similar expression in yeast and humans, have a reduced R-loop level and less DNA damage. The insertion of an intron within a gene susceptible to R-loops can also suppress the formation and recombination of R-loops. Bonnet et al. (2017) speculated that the role of introns in maintaining genetic stability may explain their evolutionary maintenance in certain locations, especially in highly expressed genes.

Individual evidence

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  8. ↑ John L. Woolford, Michael Rosbash: The use of R-looping for structural gene identification and mRNA purification . In: Nucleic Acids Research . tape 6 , no. 7 , 1979, ISSN  0305-1048 , pp. 2483–2497 , doi : 10.1093 / nar / 6.7.2483 , PMID 379820 , PMC 327867 (free full text) - ( oup.com [accessed September 22, 2019]).
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  10. ↑ T. Itoh, J. Tomizawa: Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. In: Proceedings of the National Academy of Sciences . tape 77 , no. 5 , May 1, 1980, ISSN  0027-8424 , pp. 2450–2454 , doi : 10.1073 / pnas.77.5.2450 , PMID 6156450 , PMC 349417 (free full text) - ( pnas.org [accessed September 22, 2019]).
  11. ↑ M. Drolet, X. Bi, LF Liu: Hypernegative supercoiling of the DNA template during transcription elongation in vitro . In: The Journal of Biological Chemistry . tape 269 , no. 3 , January 21, 1994, ISSN  0021-9258 , pp. 2068-2074 , PMID 8294458 .
  12. ↑ a b Matthias Groh, Natalia Gromak: Out of Balance: R-loops in Human Disease . In: PLoS Genetics . tape 10 , no. 9 , September 18, 2014, ISSN  1553-7404 , p. e1004630 , doi : 10.1371 / journal.pgen.1004630 , PMID 25233079 , PMC 4169248 (free full text) - ( plos.org [accessed on September 22, 2019]).
  13. ↑ Susana M. Cerritelli, Robert J. Crouch: Ribonuclease H: the enzymes in eukaryotes: Ribonucleases H of eukaryotes . In: FEBS Journal . tape 276 , no. 6 , March 2009, p. 1494–1505 , doi : 10.1111 / j.1742-4658.2009.06908.x , PMID 19228196 , PMC 2746905 (free full text) - ( wiley.com [accessed September 22, 2019]).
  14. ↑ Yujia A. Chan, Maria J. Aristizabal, Phoebe YT Lu, Zongli Luo, Akil Hamza: Genome-Wide Profiling of Yeast DNA: RNA Hybrid Prone Sites with DRIP-Chip . In: PLoS Genetics . tape 10 , no. 4 , April 17, 2014, ISSN  1553-7404 , p. e1004288 , doi : 10.1371 / journal.pgen.1004288 , PMID 24743342 , PMC 3990523 (free full text) - ( plos.org [accessed on September 22, 2019]).
  15. ↑ D. Roy, K. Yu, MR Lieber: Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences . In: Molecular and Cellular Biology . tape 28 , no. 1 , January 1, 2008, ISSN  0270-7306 , p. 50-60 , doi : 10.1128 / MCB.01251-07 , PMID 17954560 , PMC 2223306 (free full text) - ( asm.org [accessed September 22, 2019]).
  16. ↑ Paul A. Ginno, Paul L. Lott, Holly C. Christensen, Ian Korf, Frédéric Chédin: R-loop formation Is a Distinctive Characteristic of unmethylated CpG Human Iceland promoter . In: Molecular Cell . tape 45 , no. 6 , March 2012, p. 814–825 , doi : 10.1016 / j.molcel.2012.01.017 , PMID 22387027 , PMC 3319272 (free full text) - ( elsevier.com [accessed September 22, 2019]).
  17. ↑ Alicia D. D'Souza, Boris P. Belotserkovskii, Philip C. Hanawalt: A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system . In: Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms . tape 1861 , no. 2 , February 2018, p. 158–166 , doi : 10.1016 / j.bbagrm.2017.12.008 , PMID 29357316 , PMC 5820110 (free full text) - ( elsevier.com [accessed September 22, 2019]).
  18. ↑ Maikel Castellano-Pozo, José M. Santos-Pereira, Ana G. Rondón, Sonia Barroso, Eloisa Andújar: R Loops Are Linked to Histone H3 S10 Phosphorylation and Chromatin Condensation . In: Molecular Cell . tape 52 , no. 4 , November 2013, p. 583-590 , doi : 10.1016 / j.molcel.2013.10.006 ( elsevier.com [accessed September 22, 2019]).
  19. ^ A b Lorenzo Costantino, Douglas Koshland: The Yin and Yang of R-loop biology . In: Current Opinion in Cell Biology . tape 34 , June 2015, p. 39–45 , doi : 10.1016 / j.ceb.2015.04.008 , PMID 25938907 , PMC 4522345 (free full text) - ( elsevier.com [accessed September 22, 2019]).
  20. ↑ Boris P. Belotserkovskii, Silvia Tornaletti, Alicia D. D'Souza, Philip C. Hanawalt: R-loop generation during transcription: Formation, processing and cellular outcomes . In: DNA Repair . tape 71 , November 2018, p. 69–81 , doi : 10.1016 / j.dnarep.2018.08.009 , PMID 30190235 , PMC 6340742 (free full text) - ( elsevier.com [accessed September 22, 2019]).
  21. ↑ Julie Sollier, Karlene A. Cimprich: Breaking bad: R-loops and genome integrity . In: Trends in Cell Biology . tape 25 , no. 9 , September 2015, p. 514-522 , doi : 10.1016 / j.tcb.2015.05.003 , PMID 26045257 , PMC 4554970 (free full text) - ( elsevier.com [accessed September 22, 2019]).
  22. ↑ T. Itoh, J. Tomizawa: Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. In: Proceedings of the National Academy of Sciences . tape 77 , no. 5 , May 1, 1980, ISSN  0027-8424 , pp. 2450–2454 , doi : 10.1073 / pnas.77.5.2450 , PMID 6156450 , PMC 349417 (free full text) - ( pnas.org [accessed September 22, 2019]).
  23. ↑ a b c Amandine Bonnet, Ana R. Grosso, Abdessamad Elkaoutari, Emeline Coleno, Adrien Presle: Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability . In: Molecular Cell . tape 67 , no. 4 , August 2017, p. 608–621.e6 , doi : 10.1016 / j.molcel.2017.07.002 ( elsevier.com [accessed September 22, 2019]).