title: Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability.
abstract: R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats.
authors.affiliation: Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Kaalak
authors.initials: K
authors.lastname: Reddy
authors.affiliation: Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Monika H M
authors.initials: MH
authors.lastname: Schmidt
authors.affiliation: Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Department of Biology, Laurentian University, Sudbury, Ontario P3E 2C6, Canada.
authors.forename: Jaimie M
authors.initials: JM
authors.lastname: Geist
authors.affiliation: Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Neha P
authors.initials: NP
authors.lastname: Thakkar
authors.affiliation: Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Gagan B
authors.initials: GB
authors.lastname: Panigrahi
authors.affiliation: Department of Biochemistry & Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
authors.forename: Yuh-Hwa
authors.initials: YH
authors.lastname: Wang
authors.affiliation: Department of Genetics, The Hospital for Sick Children, Peter Gilgan Centre for Research & Learning, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 0A4, Canada cepearson.sickkids@gmail.com.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Pearson
mesh_terms: D000073885:C9orf72 Protein; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D042622:DNA Repeat Expansion; D042822:Genomic Instability; D006367:HeLa Cells; D006801:Humans; D009474:Neurons / Q000378:metabolism; D011506:Proteins / Q000235:genetics*; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D019680:Trinucleotide Repeat Expansion
chemical_list: D000073885:C9orf72 Protein; C568651:C9orf72 protein, human; D011506:Proteins; D012313:RNA; D004247:DNA; D016914:Ribonuclease H