Preferential cleavage of upstream targets in concatenated miRNA/siRNA target sites support a 5′-3′ scanning model for RISC target recognition

RNA interference (RNAi) is becoming medicine for curing human diseases. Still, we lack a thorough understanding of some fundamental aspects of RNAi that affect its efficiency and accuracy. One such question is how RNA-induced silencing complex (RISC) can efficiently find its targets. To address this question, we developed a strategy that involves the expression of mRNAs containing concatenations of identical miRNA/siRNA target sites. These mRNAs were cleaved by co-expressed miRNAs in plant cells or by co-transfected siRNAs in mammalian cells. The mRNA cleavage events were then detected using the 5′RACE assay. Using this strategy, we found that RISCs preferentially cleave the upstream ones of concatenated target sites, consistent with a model that RISC scans mRNA in 5’→3′ direction to approach its target sites. The stability of the cleaved mRNA fragments correlates with the complementarity between siRNA and its target sequence. When siRNA perfectly complements its target sequence, the cleaved mRNA fragment becomes stable and may be cleaved in a second round. Our findings have practical implications for designing siRNAs with increased efficiency and reduced off-target effects.