Reconstitution of siRNA Biogenesis In Vitro: Novel Reaction Mechanisms and RNA Channeling in the RNA-Directed DNA Methylation Pathway.

Eukaryotes deploy RNA-mediated gene silencing pathways to guard their genomes against selfish genetic elements, such as transposable elements and invading viruses. In plants, RNA-directed DNA methylation (RdDM) is used to silence selfish elements at the level of transcription. This process involves 24-nt short interfering RNAs (siRNAs) and longer noncoding RNAs to which the siRNAs base-pair. Recently, we showed that 24-nt siRNA biogenesis could be recapitulated in the test tube using purified enzymes, yielding biochemical answers to numerous questions left unresolved by prior genetic and genomic studies. Interestingly, each enzyme has activities that program what happens in the next step, thus channeling the RNAs within the RdDM pathway and restricting their diversion into alternative pathways. However, a similar mechanistic understanding is lacking for other important steps of the RdDM pathway. We discuss some of the steps most in need of biochemical investigation and important questions still in need of answers.