A Seed Mismatch Enhances Argonaute2-Catalyzed Cleavage and Partially Rescues Severely Impaired Cleavage Found in Fish

Abstract

The RNAi pathway provides both innate immunity and efficient gene-knockdown tools in many eukaryotic species, but curiously not in zebrafish. We discovered that RNAi is less effective in zebrafish at least partly because Argonaute2-catalyzed mRNA slicing is impaired. This defect is due to two mutations that arose in an ancestor of most teleost fish, implying that most fish lack effective RNAi. Despite lacking efficient slicing activity, these fish have retained the ability to produce miR-451, a microRNA generated by a cleavage reaction analogous to slicing. This ability is due to a G–G mismatch within the fish miR-451 precursor, which substantially enhances its cleavage. An analogous G–G mismatch (or sometimes also a G–A mismatch) enhances target slicing, despite disrupting seed pairing important for target binding. These results provide a strategy for restoring RNAi to zebrafish and reveal unanticipated opposing effects of a seed mismatch with implications for mechanism and guide-RNA design. Chen et al. discover two substitutions that restore normal slicing activity to zebrafish Argonaute2 and a mismatch involving guide-RNA position 6 that further enhances this activity. These findings suggest a strategy for enabling efficient RNAi in zebrafish and show that some seed mismatches have opposing effects on binding and slicing. Keywords: Argonaute; AGO2; RISC; RNAi; in vitro slicing; miRNA; miR-451 processing; siRNA design; kinetic analysis