Restoring and enhancing Argonaute2-catalyzed cleavage

Abstract

The core of the RNA silencing pathway relies on an Argonaute protein in complex with a small RNA. Together, this complex targets transcript RNAs through sequence complementarity to induce the destruction of the transcript RNA either through Argonaute2-mediated slicing or mRNA destabilization and decay. The RNAi pathway provides both innate immunity against foreign sequences, such as viruses and transposons, and has been harnessed as an efficient gene-knockdown tool 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 can be traced to two conserved mutations that arose in an ancestor of most teleost fish almost 300 million years ago, 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. This led to the surprising discovery that 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 guideƯRNA design.