Main constraints for RNAi induced by expressed long dsRNA in mouse cells

Abstract

RNA interference (RNAi) is sequence-specific mRNA degradation guided by small RNAs (siRNAs) produced from long double-stranded RNA (dsRNA) by RNase Dicer. Proteins executing RNAi are present in mammalian cells but sustain a gene-regulating microRNA pathway while dsRNA-induced innate immunity relies on a sequence-independent interferon response. While striving to benchmark mammalian RNAi analysis, we report that the main RNAi constraint is siRNA production, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of dsRNA-binding Dicer co-factors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of Protein Kinase R, a key dsRNA sensor for interferon response, had minimal positive effects in fibroblasts. Without increasing Dicer activity, RNAi can occur when the first Dicer cleavage of an abundant dsRNA produces an efficient siRNA. In mammals, efficient RNAi may effectively employ substrates, which have some features of microRNA precursors, hence bringing the two pathways mechanistically even closer. At the same time, Dicer substrate optimization, which viruses would avoid, represents an opportunity for evolving RNAi, yet unlikely as an antiviral system.