RNA stem structure governs coupling of dicing and gene silencing in RNA interference

PremicroRNAs (premiRNAs) possess secondary structures consisting of a loop and a stem with multiple mismatches. Despite the well-characterized RNAi pathway, how the structural features of premiRNA contribute to dicing and subsequent gene-silencing efficiency remains unclear. Using single-molecule FISH, we demonstrate that cytoplasmic mRNA, but not nuclear mRNA, is reduced during RNAi. The dicing rate and silencing efficiency both increase in a correlated manner as a function of the loop length. In contrast, mismatches in the stem drastically diminish the silencing efficiency without impacting the dicing rate. We show that this decoupling effect is not due to the loading to the RNA-induced silencing complex, RNA uptake, or cellular dicing. We postulate that the stem mismatches perturb the handover of the cleaved miRNAs from Dicer to Argonaute, leading to poor strand selection. Our results imply that the stem structures prevalent in cellular miRNAs have suboptimal silencing efficiency.