Reporting in The Plant Cell, Olivier Voinnet and colleagues show that viral silencing suppressors can be used to help unravel the complexity of RNA silencing.

In plants, RNA silencing is involved in the anti-viral adaptive immune response through post-transcriptional gene silencing (PTGS; known as RNA interference (RNAi) in animals). PTGS involves endonucleolytic cleavage of RNAs guided by sequence-specific small interfering RNAs (siRNAs). RNA silencing is also linked to development, in which sequence-specific microRNAs (miRNAs) either promote endonucleolytic cleavage of RNAs or inhibit the translation of target RNAs.

The similarities between the siRNA and miRNA pathways led the authors to investigate RNA silencing using the suppressor proteins that viruses use to counteract PTGS. Although viral silencing suppressors have been studied before, their mode and point of action in the PTGS pathway have remained unknown.

The authors devised an experimental system in Arabidopsis thaliana to allow the comparative side-by-side analysis of five distinct suppressors. They expressed the suppressors transgenically in the same Arabidopsis ecotype, in which PTGS of the endogenous chalcone synthase (CHS) transcript was activated.

All five suppressors inhibited PTGS of the CHS transcript, but only three altered miRNA accumulation or miRNA-guided functions. This indicates that, as in animals, the siRNA and miRNA pathways overlap only partially.

The three suppressors that altered the miRNA pathway are diverse, so the authors propose that their induction of similar developmental abnormalities might be secondary to the inhibition of the siRNA pathway. The authors also found that other Arabidopsis small silencing RNAs were resistant to all five silencing suppressors, which indicates that these RNAs have distinct biosynthetic and functional pathways. Furthermore, miRNAs were shown to fall into two size classes (as per siRNAs), and the authors suggest that 21-nucleotide miRNAs are incorporated into the RNA-induced silencing complex (RISC), whereas 24-nucleotide miRNAs might direct transcriptional silencing events.

Using this system, the authors showed that not all suppressors are equal — one partially reduces dsRNA processing by Dicer and prevents mRNA degradation, perhaps by inhibiting RISC activity, others might act downstream of Dicer, as they have no effect on dsRNA processing, and another functions by sequestering both siRNAs and miRNAs. Because one suppressor was also found to suppress RNAi in a human cell line, it is hoped that these results will ultimately help to decipher the mechanisms that underlie RNAi in animals, as well as in plants.