title: DNA-RNA hybrid formation mediates RNAi-directed heterochromatin formation.
abstract: Certain noncoding RNAs (ncRNAs) implicated in the regulation of chromatin structure associate with chromatin. During the formation of RNAi-directed heterochromatin in fission yeast, ncRNAs transcribed from heterochromatin are thought to recruit the RNAi machinery to chromatin for the formation of heterochromatin; however, the molecular details of this association are not clear. Here, using RNA immunoprecipitation assay, we showed that the heterochromatic ncRNA was associated with chromatin via the formation of a DNA-RNA hybrid and bound to the RNA-induced transcriptional silencing (RITS) complex. The presence of DNA-RNA hybrid in the cell was also confirmed by immunofluorescence analysis using anti-DNA-RNA hybrid antibody. Over-expression and depletion of RNase H in vivo decreased and increased the amount of DNA-RNA hybrid formed, respectively, and both disturbed heterochromatin. Moreover, DNA-RNA hybrid was formed on, and over-expression of RNase H inhibited the formation of, artificial heterochromatin induced by tethering of RITS to mRNA. These results indicate that heterochromatic ncRNAs are retained on chromatin via the formation of DNA-RNA hybrids and provide a platform for the RNAi-directed heterochromatin assembly and suggest that DNA-RNA hybrid formation plays a role in chromatic ncRNA function.
authors.affiliation: Laboratory of Cell Regulation and Molecular Network, Division of Systemic Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto 606-8501, Japan.
authors.forename: Mina
authors.initials: M
authors.lastname: Nakama
authors.forename: Kei
authors.initials: K
authors.lastname: Kawakami
authors.forename: Takuya
authors.initials: T
authors.lastname: Kajitani
authors.forename: Takeshi
authors.initials: T
authors.lastname: Urano
authors.forename: Yota
authors.initials: Y
authors.lastname: Murakami
mesh_terms: D047369:Chromatin Immunoprecipitation / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006570:Heterochromatin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D034622:RNA Interference; D022661:RNA, Untranslated / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism
chemical_list: D006570:Heterochromatin; D022661:RNA, Untranslated; D004247:DNA; D016914:Ribonuclease H