Biochemical Dissection of RNA Silencing in Plants

Although RNA silencing was first discovered in plants, thus far it has been studied biochemically only in animals, where itis known as RNA interference (RNAi). In animals, two components of the RNAi pathway have been identified: Dicer, a multidomainRNase III that converts long double-stranded RNA (dsRNA) into small interfering RNA (siRNA) and the RNA-induced silencingcomplex (RISC), as siRNA-containing protein–RNA complex that targets complementary mRNA for destruction. We have developedmethods for the biochemical dissection of plant RNA silencing. In this chapter, we describe in detail how to use wheat germextract to study two distinct Dicer-like activities, RNA-dependent RNA polymerase (RdRP), and endogenous microRNA-programmedRISC activities. These comprehensive protocols should prove useful in the further dissection of the plant RNA silencing pathway,as well as for the validation of the predicted targets of endogenous plant microRNAs.