Global identification of AGO3–RNA interactions inChlamydomonasreveals the small RNA–mediated regulation of nuclear and chloroplast gene expression

MicroRNAs (miRNAs) form complexes with Argonaute (AGO) proteins and bind to mRNAs with complementary sequences to repress their expression. Organisms typically possess several hundred miRNAs regulating diverse biological processes. Although the roles of miRNAs have been well elucidated in multicellular organisms, they remain largely unexplored in unicellular organisms. In the green alga Chlamydomonas reinhardtii , the first unicellular organism in which miRNAs were discovered, identifying miRNA target genes remains challenging. Here, we report the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) to generate global AGO3–RNA interaction maps, identifying 131 mRNAs derived from nuclear genes. We also detected mRNAs from two chloroplast genes, suggesting that AGO3 may also influence chloroplast gene expression. Our findings indicate that Chlamydomonas small RNAs repress nuclear and chloroplast gene expression. Furthermore, HITS-CLIP analyses are now feasible for any RNA-binding protein in Chlamydomonas , offering new opportunities to uncover the functions of RNA-binding proteins of interest.