title: Distinct and concurrent pathways of Pol II- and Pol IV-dependent siRNA biogenesis at a repetitive trans-silencer locus in Arabidopsis thaliana.
abstract: Short interfering RNAs (siRNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of target genes. In our system, siRNAs are produced by transcribing an inverted DNA repeat (IR) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into siRNAs of 21-24 nt. Primarily 24-nt siRNAs trigger RdDM of the target enhancer in trans and TGS of a downstream GFP reporter gene. We analyzed siRNA accumulation from two different structural forms of a trans-silencer locus in which tandem repeats are embedded in the enhancer IR and distinguished distinct RNA polymerase II (Pol II)- and Pol IV-dependent pathways of siRNA biogenesis. At the original silencer locus, Pol-II transcription of the IR from a 35S promoter produces a hairpin RNA that is diced into abundant siRNAs of 21-24 nt. A silencer variant lacking the 35S promoter revealed a normally masked Pol IV-dependent pathway that produces low levels of 24-nt siRNAs from the tandem repeats. Both pathways operate concurrently at the original silencer locus. siRNAs accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing siRNAs revealed the preferential accumulation of siRNAs at GC-rich regions containing methylated CG dinucleotides. In addition to supporting a correlation between base composition, DNA methylation and siRNA accumulation, our results highlight the complexity of siRNA biogenesis at repetitive loci and show that Pol II and Pol IV use different promoters to transcribe the same template.
authors.affiliation: Institute of Plant and Microbial Biology, Academia Sinica, 128, Sec. 2, Academia Road, Nankang, 115, Taipei, Taiwan; Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, Vienna, Austria.
authors.forename: Taku
authors.initials: T
authors.lastname: Sasaki
authors.forename: Tzuu-fen
authors.initials: TF
authors.lastname: Lee
authors.forename: Wen-Wei
authors.initials: WW
authors.lastname: Liao
authors.forename: Ulf
authors.initials: U
authors.lastname: Naumann
authors.forename: Jo-Ling
authors.initials: JL
authors.lastname: Liao
authors.forename: Changho
authors.initials: C
authors.lastname: Eun
authors.forename: Ya-Yi
authors.initials: YY
authors.lastname: Huang
authors.forename: Jason L
authors.initials: JL
authors.lastname: Fu
authors.forename: Pao-Yang
authors.initials: PY
authors.lastname: Chen
authors.forename: Blake C
authors.initials: BC
authors.lastname: Meyers
authors.forename: Antonius J M
authors.initials: AJ
authors.lastname: Matzke
authors.forename: Marjori
authors.initials: M
authors.lastname: Matzke
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D019175:DNA Methylation; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D017930:Genes, Reporter; D059014:High-Throughput Nucleotide Sequencing; D018519:Meristem / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D018520:Plant Shoots / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D017421:Sequence Analysis; D020080:Tandem Repeat Sequences / Q000235:genetics*
chemical_list: D029681:Arabidopsis Proteins; D018749:RNA, Plant; D034741:RNA, Small Interfering; D012319:RNA Polymerase II; C499181:RNA polymerase IV, Arabidopsis; D012321:DNA-Directed RNA Polymerases
keywords: Arabidopsis thaliana; Pol IV; RNA polymerase II; RNA-directed DNA methylation; inverted repeats; siRNAs; tandem repeats