RNA-mediated gene silencing mutants in Arabidopsis

RNA silencing involves targeted inactivation of genes by sequence interactions with homologous RNAs. The most familiar form of RNA silencing occurs at the posttranscriptional level and involves double stranded (ds) RNA- induced degradation of homologous RNAs in the cytoplasm. This phenomenon has been termed posttranscriptional gene silencing (PTGS) in plants, RNA interference (RNAi) in animals and quelling in fungi. In plants, dsRNA can also induce methylation of homologous DNA sequences (RNA-directed DNA methylation or RdDM). Our group has found that dsRNAs containing promoter sequences can induce methylation and transcriptional gene silencing (TGS) of homologous promoters in trans. This method of silencing is potentially useful in functional genomics approaches, particularly for targeting single members of gene families that have distinct promoter regions, and might play a natural role in directing epigenetic modifications to promoters of endogenous genes. Although the mechanism of PTGS/RNAi is being rapidly pieced together using genetic and biochemical approaches, the extent to which PTGS components overlap with those required for RNA-mediated TGS is not known. In addition, the ways in which RNA interacts with the DNA methylation and (perhaps) chromatin modification machineries to induce epigenetic alterations at the genome level are largely unexplored. We propose to take a genetic approach to dissect the mechanism of RNA-mediated TGS and RdDM by performing mutant screens of a promoter-dsRNA silencing system that we have established in Arabidopsis. These studies have the potential to reveal proteins that link RNA to the process of homologous DNA methylation, and to determine the degree to which shared proteins participate in both the PTGS and RNA-mediated TGS pathways.

To grow and develop properly, plants and animals need to regulate their genome so that each cell type expresses only a select subset of genes while the remaining genes are kept in a silent state. An important question concerns the way in which silencing factors are targeted to specific regions of the genome. Recent work by several labs, including our own, has shown that targeting is mediated by small RNAs generated by `dicing` longer double stranded RNA precursors in the RNA interference (RNAi) pathway. The small RNAs are able to find their matching DNA sequences in the genome, resulting in the recruitment to that site of enzymes that catalyze chemical modifications that are associated with gene silencing. Our work has focused on a variation of this type of silencing in plants called RNA-directed DNA methylation. To identify proteins required for this process, we conducted a screen to find mutant plants that are unable to carry out RNA-directed DNA methylation and silencing of a target gene. Using the model plant, Arabidopsis thaliana, we identified several novel, plant-specific proteins that we called DRD, for defective in RNA-directed DNA methylation. The DRD proteins - one is a so-called chromatin remodelling factor and the other two are subunits of a novel RNA polymerase termed Pol IV - probably help to open up the target DNA when the matching small RNA is present. This exposes the target DNA to enzymes called DNA methyltransferases, which methylate cytosines in DNA and induce gene silencing. Interestingly, we found that the DRD protein machinery might also be involved in erasing cytosine methylation, via another type of enzyme called DNA glycosylase, thus reversing silencing and reactivating target genes. We have speculated that the ability to keep target genes in a potentially reversible state of expression through the activity of the DRD proteins can help immobile plants cope with unpredictable growth conditions. Although the DRD proteins we identified are found only in the plant kingdom, evidence for a similar process of small RNA-mediated modification of chromosomes has been obtained in animals, including humans. Indeed, the feasibility of using this approach for therapeutic purposes is now being investigated by other labs. We are continuing our studies on how plants use reversible, small RNA-mediated chromosome modifications to adapt to their environment.