Heterochromatic DNA repeats in Drosophila and unusual gene silencing in yeast cells

The 1.25-kb heterochromatic Stellate repeats of Drosophila melanogaster are capable of stably persisting in transgenic constructs and silencing the white reporter gene (mosaic position effect variegation). This system reveals an unusual form of silencing, which is insensitive to known modifiers of position effect variegation. The unusual form of silencing was studied with yeast Saccharomyces cerevisiae, a simple eukaryotic model. To be transferred into yeast cells, the D. melanogaster Stellate repeats were cloned in the pYAC4 centromeric vector (CEN4, URA3, TRP1, HIS3). The HIS3 and/or URA3 genes could be inactive in plasmids consisting of pYAC4 and the Stellate insert in yeast cells. Deletion of D. melanogaster DNA from the plasmid was found to activate the URA3 and HIS3 genes. It was assumed that the genes were repressed rather than damaged in the presence of the Stellate repeats and that a new form of gene silencing was revealed in S. cerevisiae.     

The influence of inverted repeats on the production of small antisense RNAs involved in gene silencing

Gene silencing by an ACO1 [1-aminocyclopropane-1-carboxylate (ACC) oxidase 1] sense transgene in tomato plants was correlated with the production of small antisense RNAs (asRNAs) of 21-28 nucleotides, which were preferentially generated from the 3' region of the transgene. Adding inverted repeats (IRs) to the 5' untranslated region of the ACO1 transgene led to stronger silencing than was obtained with the transgene lacking the IRs, and in these plants the asRNAs were preferentially produced from the 5' region, including the IRs themselves and sequences immediately downstream. This observation indicates that secondary structure, including inverted repeats, may be a key determinant of small RNA production in gene silencing. Small asRNAs of 28 nt were much more abundant in the line containing the IRs than in the line without IRs, and may contribute to the stronger silencing associated with the IRs. Much lower levels of small RNA species were detected in plants containing an antisense ACO1 transgene than in an ACO1-sense silenced line showing weaker silencing. This suggests that the stronger suppression of the endogenous ACO1 gene by an antisense transgene may be the result of the combined effects of large antisense RNAs produced from the antisense transgene and small asRNAs.     

Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats

In a number of organisms, transgenes containing transcribed inverted repeats (IRs) that produce hairpin RNA can trigger RNA-mediated silencing, which is associated with 21-24 nucleotide small interfering RNAs (siRNAs). In plants, IR-driven RNA silencing also causes extensive cytosine methylation of homologous DNA in both the transgene "trigger" and any other homologous DNA sequences--"targets". Endogenous genomic sequences, including transposable elements and repeated elements, are also subject to RNA-mediated silencing. The RNA silencing gene ARGONAUTE4 (AGO4) is required for maintenance of DNA methylation at several endogenous loci and for the establishment of methylation at the FWA gene. Here, we show that mutation of AGO4 substantially reduces the maintenance of DNA methylation triggered by IR transgenes, but AGO4 loss-of-function does not block the initiation of DNA methylation by IRs. AGO4 primarily affects non-CG methylation of the target sequences, while the IR trigger sequences lose methylation in all sequence contexts. Finally, we find that AGO4 and the DRM methyltransferase genes are required for maintenance of siRNAs at a subset of endogenous sequences, but AGO4 is not required for the accumulation of IR-induced siRNAs or a number of endogenous siRNAs, suggesting that AGO4 may function downstream of siRNA production.