title: Detection and Characterization of R Loop Structures.
abstract: R loops are special three stranded nucleic acid structures that comprise a nascent RNA hybridized with the DNA template strand, leaving a non-template DNA single-stranded. More specifically, R loops form in vivo as G-rich RNA transcripts invade the DNA duplex and anneal to the template strand to generate an RNA:DNA hybrid, leaving the non-template, G-rich DNA strand in a largely single-stranded conformation (Aguilera and Garcia-Muse, Mol Cell 46:115-124, 2012).DNA-RNA hybrids are a natural occurrence within eukaryotic cells, with levels of these hybrids increasing at sites with high transcriptional activity, such as during transcription initiation, repression, and elongation. RNA-DNA hybrids influence genomic instability, and growing evidence points to an important role for R loops in active gene expression regulation (Ginno et al., Mol Cell 45, 814-825, 2012; Sun et al., Science 340: 619-621, 2013; Bhatia et al., Nature 511, 362-365, 2014). Analysis of the occurrence of such structures is therefore of increasing relevance and herein we describe methods for the in vivo and in vitro identification and characterization of R loops in mammalian systems.R loops (DNA:RNA hybrids and the associated single-stranded DNA) have been traditionally associated with threats to genome integrity, making some regions of the genome more prone to DNA-damaging and mutagenic agents. Initially considered to be rare byproducts of transcription, over the last decade accumulating evidence has pointed to a new view in which R loops form more frequently than previously thought. The R loop field has become an increasingly expanded area of research, placing these structures as a major threat to genome stability but also as potential regulators of gene expression. Special interest has arisen as they have also been linked to a variety of diseases, including neurological disorders and cancer, positioning them as potential therapeutic targets [5].
authors.affiliation: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Boque-Sastre
authors.affiliation: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Soler
authors.affiliation: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain.
authors.forename: Sonia
authors.initials: S
authors.lastname: Guil
mesh_terms: D018899:CpG Islands; D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D047468:Immunoprecipitation; D017404:In Situ Hybridization, Fluorescence; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
chemical_list: D012313:RNA; D004247:DNA
keywords: Bisulfite sequencing; CpG island; DRIP; In vitro transcription; R loop; RNA-FISH; Vimentin