Argonaute-CLIP delineates versatile, functional RNAi networks in Aedes aegypti, a major vector of human viruses

Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts, and they are central to RNA interference (RNAi). RNAi is critical for regulation of gene expression and antiviral defense in Aedes aegypti mosquitoes, which transmit Zika, chikungunya, dengue, and yellow fever viruses. In mosquitoes, AGO1 mediates miRNA interactions, while AGO2 mediates siRNA interactions. We applied AGO-crosslinking immunoprecipitation (AGO-CLIP) for both AGO1 and AGO2, and we developed a universal software package for CLIP analysis (CLIPflexR), identifying 230 small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map in mosquitoes. RNAi network maps predicted expression levels of small RNA targets in specific tissues. Additionally, this resource identified unexpected, context-dependent AGO2 target preferences, including endogenous viral elements and 30 UTRs. Finally, contrary to current thinking, mosquito AGO2 repressed imperfect targets. These findings expand our understanding of small RNA networks and have broad implications for the study of antiviral RNAi.

Automated summary

By using AGO-crosslinking immunoprecipitation (AGO-CLIP) for AGO1 and AGO2 in Aedes aegypti mosquitoes, researchers identified a comprehensive RNAi network map with 230 small RNAs and 5,447 small RNA targets. The study also revealed unexpected AGO2 target preferences and demonstrated that mosquito AGO2 repressed imperfect targets, expanding our understanding of small RNA networks and antiviral RNAi research.