Global identification of AGO3–RNA interactions reveals targets of small RNA-mediated gene regulation in <i>Chlamydomonas reinhardtii</i>

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 that regulate diverse aspects of biology. Although the roles of miRNAs have been elucidated in multicellular organisms, they remain largely unexplored in unicellular organisms. Identifying miRNA target genes remains challenging in the green alga Chlamydomonas (Chlamydomonas reinhardtii), the first unicellular organism in which miRNAs were discovered. Previous computational and sequencing-based approaches, such as miRNA-mRNA complementarity predictions, RNA-seq, and Ribo-seq, have struggled to identify Chlamydomonas miRNA targets. While similar technical difficulties exist in animals, crosslinking immunoprecipitation followed by sequencing has overcome these challenges. This method involves ultraviolet-mediated crosslinking of RNA-binding proteins (RBPs) to their target RNAs in living cells, followed by partial RNase digestion, immunopurification, and sequencing to map RBP-associated RNAs across the genome. Here, we performed high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) to generate a global AGO3-RNA interaction map. We identified 120 mRNAs derived from nuclear genes and two mRNAs derived from chloroplast genes. Expression levels of the nuclear gene CAS and chloroplast gene petA were higher in an ago3 mutant than in wild-type Chlamydomonas, suggesting that AGO3 represses the expression of the genes identified through HITS-CLIP analysis. Our study demonstrates that HITS-CLIP analysis is now feasible for any RBP in Chlamydomonas, offering new opportunities to uncover the functions of RBPs of interest.