Argonaute3 is a key player in miRNA-mediated target cleavage and translational repression in Chlamydomonas

Abstract

MicroRNAs (miRNAs) play important roles in diverse biological processes in eukaryotes, generally through degradation and/or inhibition of the translation of target mRNAs. MicroRNAs are loaded into Argonaute (AGO) proteins to form the RNA-induced silencing complex (RISC) and used as guides to identify complementary transcripts. The distinct functions and features, such as associated small RNA classes and modes of silencing, of individual AGO paralogs have been well documented in multicellular eukaryotes. However, this aspect of miRNA function remains poorly understood in the unicellular green alga Chlamydomonas reinhardtii, which contains three AGO paralogs. In this study, we isolated AGO2 and AGO3 insertional mutants and confirmed that AGO3 is more abundantly expressed than AGO2. MicroRNA-directed target transcript cleavage and translational repression were impaired in the AGO3 mutant background, indicating that AGO3 can mediate both modes of silencing. In contrast, although the AGO2 mutant is not a null, the involvement of AGO2 in miRNA-directed silencing appears to be more limited. Our results strongly suggest that miRNA-mediated post-transcriptional gene silencing relies primarily on AGO3 in Chlamydomonas. Significance Statement Argonaute proteins associate with different classes of small RNAs and in multicellular eukaryotes play distinct roles in gene regulation or in defense responses against transposons and viruses, but roles of Argonautes in Chlamydomonas reinhardtii are poorly studied. Here we show that miRNA-mediated post-transcriptional gene silencing in Chlamydomonas relies primarily on Argonaute3.