Endogenous miRNA in the green alga Chlamydomonas regulates gene expression through CDS-targeting

MicroRNAs (miRNA) are 21-24 nucleotide RNAs present in many eukaryotes that regulate gene expression as part of the RNA-induced silencing complex. The sequence identity of the miRNA provides the specificity to guide the silencing effector Argonaute (AGO) protein to target mRNAs via a base pairing process1. The AGO complex either promotes translation repression and/or accelerated decay of this target mRNA2. There is overwhelming evidence both in vivo and in vitro that translation repression plays a major role3–7. However, there has been controversy about which of these three mechanisms is more significant in vivo, especially when effects of miRNA on endogenous genes cannot be faithfully represented by reporter systems in which, at least in metazoans, the observed repression vastly exceeds that typically observed for endogenous mRNAs8,9. Here, we provide a comprehensive global analysis of the evolutionarily distant unicellular green alga Chlamydomonas reinhardtii to quantify the effects of miRNA on protein synthesis and RNA abundance. We show that, similar to metazoan steady-state systems, endogenous miRNAs in Chlamydomonas can regulate gene-expression both by destabilization of the mRNA and by translational repression. However, unlike metazoan miRNA where target site utilization localizes mainly to 3'UTRs, in Chlamydomonas utilized target sites lie predominantly within coding regions. These results demonstrate the evolutionarily conserved mode of action for miRNAs, but details of the mechanism diverge between plant and metazoan kingdoms.