title: A hairpin RNA construct residing in an intron efficiently triggered RNA-directed DNA methylation in tobacco.
abstract: So far, conventional hairpin RNA (hpRNA) constructs consisting of an inverted repeat (IR) of target promoters directly introduced into an expression cassette have been used to mediate de novo DNA methylation. Transcripts of such constructs resemble mRNA molecules, and are likely to be exported to the cytoplasm. The presence of hpRNAs in the cytoplasm and the nucleus may account for the simultaneous activation of post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM). We hypothesized that by retaining hpRNAs in the nucleus, efficient induction of only RdDM may be achieved. Thus, we introduced into tobacco a transgene containing an intron into which an IR of a target promoter was inserted. The intronic hpRNA initiated highly specific cis- and trans-methylation, but did not induce PTGS. No spreading of methylation into sequences flanking the region of homology between the hpRNA and the target DNA was detectable. The efficient methylation-directing activity of the intronic hpRNA may indicate a previously unrecognized role of introns, potentially regulating gene expression at the transcriptional level.
authors.affiliation: RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, 67435 Neustadt, Germany.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Dalakouras
authors.forename: Mirko
authors.initials: M
authors.lastname: Moser
authors.forename: Michèle
authors.initials: M
authors.lastname: Zwiebel
authors.forename: Gabi
authors.initials: G
authors.lastname: Krczal
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Hell
authors.forename: Michael
authors.initials: M
authors.lastname: Wassenegger
mesh_terms: D001483:Base Sequence; D019175:DNA Methylation; D049452:Green Fluorescent Proteins / Q000032:analysis; D007438:Introns / Q000502:physiology; D055029:Inverted Repeat Sequences / Q000502:physiology*; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000378:metabolism; D034622:RNA Interference; D018749:RNA, Plant / Q000502:physiology*; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes
chemical_list: D018749:RNA, Plant; D049452:Green Fluorescent Proteins