Fluorescence Correlation Spectroscopy Elucidates the Pathway of RNA Interference

Abstract

Short double-stranded RNA molecules have recently emerged as important regulators of gene expression. These small RNAs associate with a member of the Argonaute protein family in an assembly known as RNA-induced silencing complex (RISC).Here we elucidate the pathway of RNA Interference (RNAi) in vivo by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS).We show that two distinct RISC exist: a large ∼3 MDa complex in the cytoplasm and a 20-fold smaller complex in the nucleus. Nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The import of Ago2 into the nucleus is mediated by the import receptor Impotin8.We further demonstrate that FCCS can be used to study the interaction of different members of the Argonaute protein family with short double-stranded RNAs and their target mRNA molecules.