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.

RNAi途径在许多真核物种中提供了先天免疫和有效的基因敲低工具，但奇怪的是在斑马鱼中却没有。我们发现RNAi在斑马鱼中的有效性至少部分是因为Argonaute2催化的mRNA切片受损。此缺陷是由于大多数硬骨鱼的祖先出现了两个突变，这意味着大多数鱼缺乏有效的RNAi。尽管缺乏有效的切片活性，这些鱼仍保留了产生miR-451的能力，miR-451是由类似于切片的切割反应产生的microRNA。这种能力是由于鱼类miR-451前体中的GG不匹配，从而大大增强了其裂解能力。尽管破坏了对目标结合很重要的种子配对，但类似的G–G不匹配（有时也可能是G–A不匹配）会增强目标切片。