RNAiMediated TGS

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Double-stranded RNAs can also produce TGS of homologous genomic regions (regions complementary to the siRNAs) in Arabidopsis, Schizosaccharomyces pombe, Drosophila, and mammalian cells (reviewed in Matzke and Birchler 2005). TGS was first observed when doubly transformed tobacco plants surprisingly exhibited a suppressed phenotype of a transgene. Closer examination indicated that observed suppression of the transgene was the result of directed DNA methylation at the transgene loci (Matzke et al. 1989). As it turned out the observed TGS in plants was mediated by dsRNAs, which was substantiated in viroid-infected plants (Wassenegger et al. 1994) and shown to be the result of RNA-dependent DNA methylation (RdDM). The action of RdDM requires a dsRNA that is subsequently processed to yield short RNAs (Wassenegger et al. 1994; Mette et al. 2000). Interestingly, it was these short dsRNAs in the doubly transformed tobacco plant that happened to include sequences that were identical to genomic promoter regions involved in the transgene expression and ultimately led to TGS via methylation of

Fig. 1 Post-transcriptional vs transcriptional gene silencing. Synthetic siRNAs transfected directly into the cell can modulate not only post-transcriptional gene silencing (PTGS) via Dicer and RISC but also direct epigenetic modifications to the corresponding genomic loci (the DNA that codes for the targeted mRNA). This nuclear component appears to involve Ago-1. To direct PTGS, the transfected siRNA is processed by Dicer/TRBP/PACT (1) and then enters into the Ago-2-containing RISC complex (2) where the target mRNA is sliced by the action of Ago-2 (3). Alternatively, synthetic siRNAs can localize to the nucleus in an Ago-1 dependent manner (A) and specifically target the homologous genomic sequence to induce chromatin modifications such as H3K9me2+ or H3K27me3+ (B) which are known to result in the conversion of euchromatin to hetero-chromatin and subsequent transcriptional gene silencing

Fig. 1 Post-transcriptional vs transcriptional gene silencing. Synthetic siRNAs transfected directly into the cell can modulate not only post-transcriptional gene silencing (PTGS) via Dicer and RISC but also direct epigenetic modifications to the corresponding genomic loci (the DNA that codes for the targeted mRNA). This nuclear component appears to involve Ago-1. To direct PTGS, the transfected siRNA is processed by Dicer/TRBP/PACT (1) and then enters into the Ago-2-containing RISC complex (2) where the target mRNA is sliced by the action of Ago-2 (3). Alternatively, synthetic siRNAs can localize to the nucleus in an Ago-1 dependent manner (A) and specifically target the homologous genomic sequence to induce chromatin modifications such as H3K9me2+ or H3K27me3+ (B) which are known to result in the conversion of euchromatin to hetero-chromatin and subsequent transcriptional gene silencing the homologous promoter and the observed reduction in transgene expression (Matzke et al. 1989). In general, transcriptional gene silencing in plants is carried out by a larger size class of siRNAs, 24-26 nucleotides (nt) in length (Hamilton et al. 2002; Zilberman et al. 2003).

Recently members of the Argonaute protein family in Arabidopsis have been shown to play an essential role in RdDM of promoter DNA and transposon silencing (Lippman et al. 2003). Specifically, Ago-4 is known to direct siRNA-mediated silencing, and Ago-4 mutants display reactivation of silent SUP alleles, along with a corresponding decrease in both CpNpG DNA and H3K9 methylation (Zilberman et al. 2003). Consequently, in plants, siRNAs that include sequences with homology to genomic promoter regions are capable of directing the methylation of the homologous promoter and subsequent transcriptional gene silencing.

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