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

RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.