RNA-directed DNA methylation mediated by DRD1 and Pol IVb: a versatile pathway for transcriptional gene silencing in plants

RNA-directed DNA methylation, which is one of several RNAi-mediated pathways in the nucleus, has been highly elaborated in the plant kingdom. RNA-directed DNA methylation requires for the most part conventional DNA methyltransferases, histone modifying enzymes and RNAi proteins; however, several novel, plant-specific proteins that are essential for this process have been identified recently. DRD1 (defective in RNA-directed DNA methylation) is a putative SWI2/SNF2-like chromatin remodelling protein; DRD2 and DRD3 (renamed NRPD2a and NRPD1b, respectively) are subunits of Pol IVb, a putative RNA polymerase found only in plants. Interestingly, DRD1 and Pol IVb appear to be required not only for RNA-directed de novo methylation, but also for full erasure of methylation when the RNA trigger is withdrawn. These proteins thus have the potential to facilitate dynamic regulation of DNA methylation. Prominent targets of RNA-directed DNA methylation in the Arabidopsis thaliana genome include retrotransposon long terminal repeats (LTRs), which have bidirectional promoter/enhancer activities, and other types of intergenic transposons and repeats. Intergenic solitary LTRs that are targeted for reversible methylation by the DRD1/Pol IVb pathway can potentially act as switches or rheostats for neighboring plant genes. The resulting alterations in gene expression patterns may promote physiological flexibility and adaptation to the environment.     

MicroRNAs guide asymmetric DNA modifications guiding asymmetric organs

In plants and animals, microRNAs have been shown to regulate target genes by inhibiting translation or altering target mRNA stability. In this issue of Developmental Cell, Bao et al. extend the known mechanisms of action of microRNAs to RNA-directed DNA methylation, a mechanism previously associated only with siRNA-mediated gene silencing.     

Two-step recruitment of RNA-directed DNA methylation to tandem repeats

Tandem repeat sequences are frequently associated with gene silencing phenomena. The Arabidopsis thaliana FWA gene contains two tandem repeats and is an efficient target for RNA-directed de novo DNA methylation when it is transformed into plants. We showed that the FWA tandem repeats are necessary and sufficient for de novo DNA methylation and that repeated character rather than intrinsic sequence is likely important. Endogenous FWA can adopt either of two stable epigenetic states: methylated and silenced or unmethylated and active. Surprisingly, we found small interfering RNAs (siRNAs) associated with FWA in both states. Despite this, only the methylated form of endogenous FWA could recruit further RNA-directed DNA methylation or cause efficient de novo methylation of transgenic FWA. This suggests that RNA-directed DNA methylation occurs in two steps: first, the initial recruitment of the siRNA-producing machinery, and second, siRNA-directed DNA methylation either in cis or in trans. The efficiency of this second step varies depending on the nature of the siRNA-producing locus, and at some loci, it may require pre-existing chromatin modifications such as DNA methylation itself. Enhancement of RNA-directed DNA methylation by pre-existing DNA methylation could create a self-reinforcing system to enhance the stability of silencing. Tandem repeats throughout the Arabidopsis genome produce siRNAs, suggesting that repeat acquisition may be a general mechanism for the evolution of gene silencing.     

Training molecularly enabled field biologists to understand organism-level gene function

A gene's influence on an organism's Darwinian fitness ultimately determines whether it will be lost, maintained or modified by natural selection, yet biologists have few gene expression systems in which to measure whole-organism gene function. In the Department of Molecular Ecology at the Max Planck Institute for Chemical Ecology we are training "molecularly enabled field biologists" to use transformed plants silenced in the expression of environmentally regulated genes and the plant's native habitats as "laboratories." Research done in these natural laboratories will, we hope, increase our understanding of the function of genes at the level of the organism. Examples of the role of threonine deaminase and RNA-directed RNA polymerases illustrate the process.     

Roles of RNA polymerase IV in gene silencing

Eukaryotes typically have three multi-subunit enzymes that decode the nuclear genome into RNA: DNA-dependent RNA polymerases I, II and III (Pol I, II and III). Remarkably, higher plants have five multi-subunit nuclear RNA polymerases: the ubiquitous Pol I, II and III, which are essential for viability; plus two non-essential polymerases, Pol IVa and Pol IVb, which specialize in small RNA-mediated gene silencing pathways. There are numerous examples of phenomena that require Pol IVa and/or Pol IVb, including RNA-directed DNA methylation of endogenous repetitive elements, silencing of transgenes, regulation of flowering-time genes, inducible regulation of adjacent gene pairs, and spreading of mobile silencing signals. Although biochemical details concerning Pol IV enzymatic activities are lacking, genetic evidence suggests several alternative models for how Pol IV might function.     

RNA silencing and plant viral diseases

RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.