Ultra-deep Coverage Single-molecule R-loop Footprinting Reveals Principles of R-loop Formation.
DNA:RNA immunoprecipitation
Nondenaturing bisulfite conversion
R-loops
S9.6 antibody
SMRT-sequencing
Journal
Journal of molecular biology
ISSN: 1089-8638
Titre abrégé: J Mol Biol
Pays: Netherlands
ID NLM: 2985088R
Informations de publication
Date de publication:
27 03 2020
27 03 2020
Historique:
received:
22
08
2019
revised:
27
01
2020
accepted:
06
02
2020
pubmed:
28
2
2020
medline:
28
8
2020
entrez:
28
2
2020
Statut:
ppublish
Résumé
R-loops are a prevalent class of non-B DNA structures that have been associated with both positive and negative cellular outcomes. DNA:RNA immunoprecipitation (DRIP) approaches based on the anti-DNA:RNA hybrid S9.6 antibody revealed that R-loops form dynamically over conserved genic hotspots. We have developed an orthogonal approach that queries R-loops via the presence of long stretches of single-stranded DNA on their looped-out strand. Nondenaturing sodium bisulfite treatment catalyzes the conversion of unpaired cytosines to uracils, creating permanent genetic tags for the position of an R-loop. Long-read, single-molecule PacBio sequencing allows the identification of R-loop 'footprints' at near nucleotide resolution in a strand-specific manner on long single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting coupled with PacBio sequencing (SMRF-seq) revealed a strong agreement between S9.6-based and bisulfite-based R-loop mapping and confirmed that R-loops form over genic hotspots, including gene bodies and terminal gene regions. Based on the largest single-molecule R-loop dataset to date, we show that individual R-loops form nonrandomly, defining discrete sets of overlapping molecular clusters that pileup through larger R-loop zones. R-loops most often map to intronic regions and their individual start and stop positions do not match with intron-exon boundaries, reinforcing the model that they form cotranscriptionally from unspliced transcripts. SMRF-seq further established that R-loop distribution patterns are not simply driven by intrinsic DNA sequence features but most likely also reflect DNA topological constraints. Overall, DRIP-based and SMRF-based approaches independently provide a complementary and congruent view of R-loop distribution, consolidating our understanding of the principles underlying R-loop formation.
Identifiants
pubmed: 32105733
pii: S0022-2836(20)30164-9
doi: 10.1016/j.jmb.2020.02.014
pmc: PMC7669280
mid: NIHMS1568681
pii:
doi:
Substances chimiques
RNA
63231-63-0
DNA
9007-49-2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
2271-2288Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM120607
Pays : United States
Organisme : NIGMS NIH HHS
ID : T32 GM008799
Pays : United States
Informations de copyright
Copyright © 2020 Elsevier Ltd. All rights reserved.
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