Deconvolution of seed and RNA-binding protein crosstalk in RNAi-based functional genomics

RNA interference (RNAi) is a major powerful platform for gene perturbations, but is restricted by off-target mechanisms. Communication between RNAs, small RNAs, and RNA-binding proteins (RBPs) is a pervasive feature of cellular RNA networks. We present a new crosstalk scenario, designated as “crosstalk with endogenous RBPs (ceRBP)”, where siRNAs or miRNAs with seed sequences that overlap RBP motifs have extended biological effects by perturbing endogenous RBP activity. Systematic analysis of siRNA off-target data and genome-wide RNAi cancer lethality screens using 501 human cancer cell lines, a cancer dependency map, revealed that seed-to-RBP crosstalk is widespread, contributes to off-target activity, and affects RNAi performance. Specifically, deconvolution of the interactions between gene knockdown and seed-mediated silencing effects in the cancer dependency map showed widespread contributions of seed-to-RBP crosstalk to growth-phenotype modulation. These findings suggest a novel aspect of miRNA biology and offer a basis for improvement of RNAi agents and RNAi-based functional genomics.