Distinct roles of Argonaute in the green alga Chlamydomonas reveal evolutionary conserved mode of miRNA-mediated gene expression

Betty Y.-W Chung,Katherine Brown,Silvia Hnatova,Francisco J Navarro,Michael J Deery,Attila Molnar,Adrian Valli,Julie Howard,David C Baulcombe

doi:10.1038/s41598-019-47415-x

Betty Y.-W Chung, Katherine Brown + Show 7 more

Open Access

https://doi.org/10.1038/s41598-019-47415-x

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Abstract

The unicellular green alga Chlamydomonas reinhardtii is evolutionarily divergent from higher plants, but has a fully functional silencing machinery including microRNA (miRNA)-mediated translation repression and mRNA turnover. However, distinct from the metazoan machinery, repression of gene expression is primarily associated with target sites within coding sequences instead of 3′UTRs. This feature indicates that the miRNA-Argonaute (AGO) machinery is ancient and the primary function is for post transcriptional gene repression and intermediate between the mechanisms in the rest of the plant and animal kingdoms. Here, we characterize AGO2 and 3 in Chlamydomonas, and show that cytoplasmically enriched Cr-AGO3 is responsible for endogenous miRNA-mediated gene repression. Under steady state, mid-log phase conditions, Cr-AGO3 binds predominantly miR-C89, which we previously identified as the predominant miRNA with effects on both translation repression and mRNA turnover. In contrast, the paralogue Cr-AGO2 is nuclear enriched and exclusively binds to 21-nt siRNAs. Further analysis of the highly similar Cr-AGO2 and Cr-AGO 3 sequences (90% amino acid identity) revealed a glycine-arginine rich N-terminal extension of ~100 amino acids that, given previous work on unicellular protists, may associate AGO with the translation machinery. Phylogenetic analysis revealed that this glycine-arginine rich N-terminal extension is present outside the animal kingdom and is highly conserved, consistent with our previous proposal that miRNA-mediated CDS-targeting operates in this green alga.

Highlights

The diverse natural roles for RNA-silencing in eukaryotes range from defense against viruses to the regulation of gene expression and chromatin remodeling[1,2,3,4,5,6,7,8,9]
The haploid unicellular green alga Chlamydomonas reinhardtii has a complex set of small RNAs and diverse RNA silencing pathways ranging from miRNA-mediated cleavage of target mRNA to translational repression[24,25,26,27,28,29]
AGO1 has a likely role in genome defense against transposable elements[30], while Cr-AGO3 is probably responsible for miRNA-mediated transcript cleavage and translational repression[31]

Summary

Introduction

The diverse natural roles for RNA-silencing in eukaryotes range from defense against viruses to the regulation of gene expression and chromatin remodeling[1,2,3,4,5,6,7,8,9]. To further characterize the Cr-AGO proteins responsible for miRNA- or siRNA-mediated gene expression, we raised antibodies against peptides from the PAZ and PIWI domains for both Cr-AGO2 and 3 (Fig. 2a), and used them for both western blot and immunoprecipitation.

Results

Conclusion