OpenPlant PI David Baulcombe and colleagues recently published two papers: (1) on the miRNA-Argonaute machinery in the unicellular green alga Chlamydomonas reinhardtii, and (2) on the application of miRNAs for regulation of synthetic gene systems in this organism:
Distinct roles of Argonaute in the green alga Chlamydomonas reveal evolutionary conserved mode of miRNA-mediated gene expression
Betty Y.-W. Chung, Adrian Valli, Michael J. Deery, Francisco J. Navarro, Katherine Brown, Silvia Hnatova, Julie Howard, Attila Molnar & David C. Baulcombe
Sci Rep. 2019; 9: 11091. doi: 10.1038/s41598-019-47415-x
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 identifed as the predominant miRNA with efects 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.
miRNA-mediated regulation of synthetic gene circuits in the green alga Chlamydomonas reinhardtii
Francisco J. Navarro and David C. Baulcombe
ACS Synth Biol. 2019 February 15; 8(2): 358–370. doi:10.1021/acssynbio.8b00393.
microRNAs (miRNAs), small RNA molecules of 20–24 nts, have many features that make them useful tools for gene expression regulation — small size, flexible design, target predictability and action at a late stage of the gene expression pipeline. In addition, their role in fine-tuning gene expression can be harnessed to increase robustness of synthetic gene networks. In this work we apply a synthetic biology approach to characterize miRNA-mediated gene expression regulation in the unicellular green alga Chlamydomonas reinhardtii. This characterization is then used to build tools based on miRNAs, such as synthetic miRNAs, miRNA-responsive 3’UTRs, miRNA decoys and self-regulatory loops. These tools will facilitate the engineering of gene expression for new applications and improved traits in this alga.