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词条 R-loop
释义

  1. History

  2. R-loop mapping

  3. R-loops in vivo

     Regulation of R-loop formation and resolution  Roles of R-loops in genetic regulation  R-loops as genetic damage 

  4. R-loops, Introns and DNA damage

  5. See also

  6. References

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.

History

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.[7]

R-loop mapping

R-loop mapping is a laboratory technique used to distinguish introns from exons in double-stranded DNA.[8] These R-loops are visualized by electron microscopy and reveal intron regions of DNA by creating unbound loops at these regions.[9]

R-loops in vivo

The potential for R-loops to serve as replication primers was demonstrated in 1980.[10] In 1994, R-loops were demonstrated to be present in vivo through analysis of plasmids isolated from E. coli mutants carrying mutations in topoisomerase.[11] 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.[12]

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.[13] 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.[14]

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.[15] They also appear to play a role in protecting some active promoters from methylation.[16] The presence of R-loops can also inhibit transcription.[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.[20] 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.[21] As well, R-loops may induce unscheduled replication by acting as a primer.[10][19]

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.[12]

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 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.[22] Genome-wide analysis showed that intron-containing genes display decreased R-loop levels and decreased DNA damage compared to intronless genes of similar expression in both yeast and humans.[22] Inserting an intron within an R-loop prone gene can also suppress R-loop formation and recombination. Bonnet et al. (2017)[22] speculated that the function of introns in maintaining genetic stability may explain their evolutionary maintenance at certain locations, particularly in highly expressed genes.

See also

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

References

1. ^{{cite journal | vauthors = Thomas M, White RL, Davis RW | title = Hybridization of RNA to double-stranded DNA: formation of R-loops | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 73 | issue = 7 | pages = 2294–8 | date = July 1976 | pmid = 781674 | pmc = 430535 | doi = 10.1073/pnas.73.7.2294 | bibcode = 1976PNAS...73.2294T }}
2. ^1 {{cite journal | vauthors = Berget SM, Moore C, Sharp PA | title = Spliced segments at the 5' terminus of adenovirus 2 late mRNA | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 74 | issue = 8 | pages = 3171–5 | date = August 1977 | pmid = 269380 | pmc = 431482 | doi = 10.1073/pnas.74.8.3171 | bibcode = 1977PNAS...74.3171B }}
3. ^{{cite journal | vauthors = Chow LT, Gelinas RE, Broker TR, Roberts RJ | title = An amazing sequence arrangement at the 5' ends of adenovirus 2 messenger RNA | journal = Cell | volume = 12 | issue = 1 | pages = 1–8 | date = September 1977 | pmid = 902310 | doi = 10.1016/0092-8674(77)90180-5 }}
4. ^{{cite journal | vauthors = Lai EC, Woo SL, Dugaiczyk A, Catterall JF, O'Malley BW | title = The ovalbumin gene: structural sequences in native chicken DNA are not contiguous | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 75 | issue = 5 | pages = 2205–9 | date = May 1978 | pmid = 276861 | pmc = 392520 | doi = 10.1073/pnas.75.5.2205 | bibcode = 1978PNAS...75.2205L }}
5. ^{{cite journal | vauthors = O'Hare K, Breathnach R, Benoist C, Chambon P | title = No more than seven interruptions in the ovalbumin gene: comparison of genomic and double-stranded cDNA sequences | journal = Nucleic Acids Research | volume = 7 | issue = 2 | pages = 321–34 | date = September 1979 | pmid = 493147 | pmc = 328020 | doi = 10.1093/nar/7.2.321 }}
6. ^{{cite journal | vauthors = Cech TR, Rio DC | title = Localization of transcribed regions on extrachromosomal ribosomal RNA genes of Tetrahymena thermophila by R-loop mapping | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 76 | issue = 10 | pages = 5051–5 | date = October 1979 | pmid = 291921 | pmc = 413077 | doi = 10.1073/pnas.76.10.5051 | bibcode = 1979PNAS...76.5051C }}
7. ^{{cite journal | vauthors = Boguslawski SJ, Smith DE, Michalak MA, Mickelson KE, Yehle CO, Patterson WL, Carrico RJ | title = Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids | journal = Journal of Immunological Methods | volume = 89 | issue = 1 | pages = 123–30 | date = May 1986 | pmid = 2422282 | doi = 10.1016/0022-1759(86)90040-2 }}
8. ^{{cite journal | vauthors = Woolford JL, Rosbash M | title = The use of R-looping for structural gene identification and mRNA purification | journal = Nucleic Acids Research | volume = 6 | issue = 7 | pages = 2483–97 | date = June 1979 | pmid = 379820 | pmc = 327867 | doi = 10.1093/nar/6.7.2483 }}
9. ^King RC, Stansfield WD, Mulligan PK (2007). A Dictionary of Genetics. Oxford University Press 7.
10. ^1 {{cite journal | vauthors = Itoh T, Tomizawa J | title = Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 77 | issue = 5 | pages = 2450–4 | date = May 1980 | pmid = 6156450 | pmc = 349417 | doi = 10.1073/pnas.77.5.2450 | bibcode = 1980PNAS...77.2450I }}
11. ^{{cite journal | vauthors = Drolet M, Bi X, Liu LF | title = Hypernegative supercoiling of the DNA template during transcription elongation in vitro | journal = The Journal of Biological Chemistry | volume = 269 | issue = 3 | pages = 2068–74 | date = January 1994 | pmid = 8294458 }}
12. ^1 {{cite journal | vauthors = Groh M, Gromak N | title = Out of balance: R-loops in human disease | journal = PLoS Genetics | volume = 10 | issue = 9 | pages = e1004630 | date = September 2014 | pmid = 25233079 | pmc = 4169248 | doi = 10.1371/journal.pgen.1004630 }}
13. ^{{cite journal | vauthors = Cerritelli SM, Crouch RJ | title = Ribonuclease H: the enzymes in eukaryotes | journal = The FEBS Journal | volume = 276 | issue = 6 | pages = 1494–505 | date = March 2009 | pmid = 19228196 | pmc = 2746905 | doi = 10.1111/j.1742-4658.2009.06908.x }}
14. ^{{cite journal | vauthors = Chan YA, Aristizabal MJ, Lu PY, Luo Z, Hamza A, Kobor MS, Stirling PC, Hieter P | title = Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip | journal = PLoS Genetics | volume = 10 | issue = 4 | pages = e1004288 | date = April 2014 | pmid = 24743342 | pmc = 3990523 | doi = 10.1371/journal.pgen.1004288 }}
15. ^{{cite journal | vauthors = Roy D, Yu K, Lieber MR | title = Mechanism of R-loop formation at immunoglobulin class switch sequences | journal = Molecular and Cellular Biology | volume = 28 | issue = 1 | pages = 50–60 | date = January 2008 | pmid = 17954560 | pmc = 2223306 | doi = 10.1128/mcb.01251-07 }}
16. ^{{cite journal | vauthors = Ginno PA, Lott PL, Christensen HC, Korf I, Chédin F | title = R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters | journal = Molecular Cell | volume = 45 | issue = 6 | pages = 814–25 | date = March 2012 | pmid = 22387027 | pmc = 3319272 | doi = 10.1016/j.molcel.2012.01.017 }}
17. ^{{cite journal | vauthors = D'Souza AD, Belotserkovskii BP, Hanawalt PC | title = A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system | journal = Biochimica et Biophysica Acta. Gene Regulatory Mechanisms | volume = 1861 | issue = 2 | pages = 158–166 | date = February 2018 | pmid = 29357316 | pmc = 5820110 | doi = 10.1016/j.bbagrm.2017.12.008 }}
18. ^{{cite journal | vauthors = Castellano-Pozo M, Santos-Pereira JM, Rondón AG, Barroso S, Andújar E, Pérez-Alegre M, García-Muse T, Aguilera A | title = R loops are linked to histone H3 S10 phosphorylation and chromatin condensation | journal = Molecular Cell | volume = 52 | issue = 4 | pages = 583–90 | date = November 2013 | pmid = 24211264 | doi = 10.1016/j.molcel.2013.10.006 }}
19. ^1 {{cite journal | vauthors = Costantino L, Koshland D | title = The Yin and Yang of R-loop biology | journal = Current Opinion in Cell Biology | volume = 34 | issue = | pages = 39–45 | date = June 2015 | pmid = 25938907 | pmc = 4522345 | doi = 10.1016/j.ceb.2015.04.008 }}
20. ^{{cite journal | vauthors = Belotserkovskii BP, Tornaletti S, D'Souza AD, Hanawalt PC | title = R-loop generation during transcription: Formation, processing and cellular outcomes | journal = DNA Repair | volume = 71 | pages = 69–81 | date = November 2018 | pmid = 30190235 | pmc = 6340742 | doi = 10.1016/j.dnarep.2018.08.009 }}
21. ^{{cite journal | vauthors = Sollier J, Cimprich KA | title = Breaking bad: R-loops and genome integrity | journal = Trends in Cell Biology | volume = 25 | issue = 9 | pages = 514–22 | date = September 2015 | pmid = 26045257 | pmc = 4554970 | doi = 10.1016/j.tcb.2015.05.003 }}
22. ^1 2 {{cite journal | vauthors = Bonnet A, Grosso AR, Elkaoutari A, Coleno E, Presle A, Sridhara SC, Janbon G, Géli V, de Almeida SF, Palancade B | title = Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability | journal = Molecular Cell | volume = 67 | issue = 4 | pages = 608–621.e6 | date = August 2017 | pmid = 28757210 | doi = 10.1016/j.molcel.2017.07.002 }}

2 : DNA|RNA splicing
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