title: R-loop-mediated genome instability in mRNA cleavage and polyadenylation mutants.
abstract: Genome instability via RNA:DNA hybrid-mediated R loops has been observed in mutants involved in various aspects of transcription and RNA processing. The prevalence of this mechanism among essential chromosome instability (CIN) genes remains unclear. In a secondary screen for increased Rad52 foci in CIN mutants, representing ∼25% of essential genes, we identified seven essential subunits of the mRNA cleavage and polyadenylation (mCP) machinery. Genome-wide analysis of fragile sites by chromatin immunoprecipitation (ChIP) and microarray (ChIP-chip) of phosphorylated H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of R loops. In parallel, we directly detected increased RNA:DNA hybrid formation in mCP mutants and demonstrated that CIN is suppressed by expression of the R-loop-degrading enzyme RNaseH. To investigate the conservation of CIN in mCP mutants, we focused on FIP1L1, the human ortholog of yeast FIP1, a conserved mCP component that is part of an oncogenic fusion in eosinophilic leukemia. We found that truncation fusions of yeast FIP1 analogous to those in cancer cause loss of function and that siRNA knockdown of FIP1L1 in human cells increases DNA damage and chromosome breakage. Our findings illuminate how mCP maintains genome integrity by suppressing R-loop formation and suggest that this function may be relevant to certain human cancers.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Stirling
authors.forename: Yujia A
authors.initials: YA
authors.lastname: Chan
authors.forename: Sean W
authors.initials: SW
authors.lastname: Minaker
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Aristizabal
authors.forename: Irene
authors.initials: I
authors.lastname: Barrett
authors.forename: Payal
authors.initials: P
authors.lastname: Sipahimalani
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kobor
authors.forename: Philip
authors.initials: P
authors.lastname: Hieter
mesh_terms: D043283:Chromosome Fragile Sites; D042822:Genomic Instability / Q000235:genetics*; D045325:HCT116 Cells; D006801:Humans; D009154:Mutation; D016366:Open Reading Frames; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: C450579:FIP1 protein, S cerevisiae; C479818:FIP1L1 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors