R-loop

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Schematic representation of factors promoting R-loop formation and stabilization R-loop promoting factors.jpg
Schematic representation of factors promoting R-loop formation and stabilization

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.

Contents

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

An illustration showing how a DNA-mRNA hybrid forms R-Loops in the regions where introns have been removed through splicing exons. R loop.jpg
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. [2] 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. [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 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] 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]

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. [13]

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. [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.

See also

Related Research Articles

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<span class="mw-page-title-main">Histone</span> Family proteins package and order the DNA into structural units called nucleosomes.

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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 term intron refers to both the DNA sequence within a gene and the corresponding RNA sequence in RNA transcripts. The non-intron sequences that become joined by this RNA processing to form the mature RNA are called exons.

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<span class="mw-page-title-main">Transcription (biology)</span> Process of copying a segment of DNA into RNA

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A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs (snRNA) and numerous proteins. Small nuclear RNA (snRNA) molecules bind to specific proteins to form a small nuclear ribonucleoprotein complex, which in turn combines with other snRNPs to form a large ribonucleoprotein complex called a spliceosome. The spliceosome removes introns from a transcribed pre-mRNA, a type of primary transcript. This process is generally referred to as splicing. An analogy is a film editor, who selectively cuts out irrelevant or incorrect material from the initial film and sends the cleaned-up version to the director for the final cut.

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<span class="mw-page-title-main">Primary transcript</span> RNA produced by transcription

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<span class="mw-page-title-main">Triple-stranded DNA</span> DNA structure

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<span class="mw-page-title-main">Eukaryotic transcription</span> Transcription is heterocatalytic function of DNA

Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of transportable complementary RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells. Unlike prokaryotic RNA polymerase that initiates the transcription of all different types of RNA, RNA polymerase in eukaryotes comes in three variations, each translating a different type of gene. A eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures. The complexity of the eukaryotic genome necessitates a great variety and complexity of gene expression control.

<span class="mw-page-title-main">Intrinsic termination</span>

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References

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  5. ↑ Lai EC, Woo SL, Dugaiczyk A, Catterall JF, O'Malley BW (May 1978). "The ovalbumin gene: structural sequences in native chicken DNA are not contiguous". Proceedings of the National Academy of Sciences of the United States of America. 75 (5): 2205–9. Bibcode:1978PNAS...75.2205L. doi: 10.1073/pnas.75.5.2205 . PMC   392520 . PMID   276861.
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  12. ↑ Drolet M, Bi X, Liu LF (January 1994). "Hypernegative supercoiling of the DNA template during transcription elongation in vitro". The Journal of Biological Chemistry. 269 (3): 2068–74. doi: 10.1016/S0021-9258(17)42136-3 . PMID   8294458.
  13. 1 2 Groh M, Gromak N (September 2014). "Out of balance: R-loops in human disease". PLOS Genetics. 10 (9): e1004630. doi:10.1371/journal.pgen.1004630. PMC   4169248 . PMID   25233079.
  14. ↑ Cerritelli SM, Crouch RJ (March 2009). "Ribonuclease H: the enzymes in eukaryotes". The FEBS Journal. 276 (6): 1494–505. doi:10.1111/j.1742-4658.2009.06908.x. PMC   2746905 . PMID   19228196.
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  16. ↑ Roy D, Yu K, Lieber MR (January 2008). "Mechanism of R-loop formation at immunoglobulin class switch sequences". Molecular and Cellular Biology. 28 (1): 50–60. doi:10.1128/mcb.01251-07. PMC   2223306 . PMID   17954560.
  17. ↑ Ginno PA, Lott PL, Christensen HC, Korf I, Chédin F (March 2012). "R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters". Molecular Cell. 45 (6): 814–25. doi:10.1016/j.molcel.2012.01.017. PMC   3319272 . PMID   22387027.
  18. ↑ D'Souza AD, Belotserkovskii BP, Hanawalt PC (February 2018). "A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system". Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1861 (2): 158–166. doi:10.1016/j.bbagrm.2017.12.008. PMC   5820110 . PMID   29357316.
  19. ↑ Castellano-Pozo M, Santos-Pereira JM, Rondón AG, Barroso S, Andújar E, Pérez-Alegre M, García-Muse T, Aguilera A (November 2013). "R loops are linked to histone H3 S10 phosphorylation and chromatin condensation". Molecular Cell. 52 (4): 583–90. doi: 10.1016/j.molcel.2013.10.006 . PMID   24211264.
  20. 1 2 Costantino L, Koshland D (June 2015). "The Yin and Yang of R-loop biology". Current Opinion in Cell Biology. 34: 39–45. doi:10.1016/j.ceb.2015.04.008. PMC   4522345 . PMID   25938907.
  21. ↑ Belotserkovskii BP, Tornaletti S, D'Souza AD, Hanawalt PC (November 2018). "R-loop generation during transcription: Formation, processing and cellular outcomes". DNA Repair. 71: 69–81. doi:10.1016/j.dnarep.2018.08.009. PMC   6340742 . PMID   30190235.
  22. ↑ Sollier J, Cimprich KA (September 2015). "Breaking bad: R-loops and genome integrity". Trends in Cell Biology. 25 (9): 514–22. doi:10.1016/j.tcb.2015.05.003. PMC   4554970 . PMID   26045257.
  23. 1 2 3 Bonnet A, Grosso AR, Elkaoutari A, Coleno E, Presle A, Sridhara SC, Janbon G, Géli V, de Almeida SF, Palancade B (August 2017). "Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability". Molecular Cell. 67 (4): 608–621.e6. doi: 10.1016/j.molcel.2017.07.002 . PMID   28757210.
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