Identification of AGO3-associated miRNAs and computational prediction of their targets in the green alga Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii harbors many types of small RNAs (sRNAs) but little is known about their role(s) in the regulation of endogenous genes and cellular processes. To define functional microRNAs (miRNAs) in Chlamydomonas, we characterized sRNAs associated with an argonaute protein, AGO3, by affinity purification and deep sequencing. Using a stringent set of criteria for canonical miRNA annotation, we identified 39 precursor miRNAs, which produce 45 unique, AGO3- associated miRNA sequences including 13 previously reported miRNAs and 32 novel ones. Potential miRNA targets were identified based on the complementarity of miRNAs with candidate binding sites on transcripts and classified, depending on the extent of complementarity, as being likely to be regulated through cleavage or translational repression. The search for cleavage targets identified 74 transcripts. However, only 6 of them showed an increase in messenger RNA (mRNA) levels in a mutant strain almost devoid of sRNAs. The search for translational repression targets, which used complementarity criteria more stringent than those empirically required for a reduction in target protein levels, identified 488 transcripts. However, unlike observations in metazoans, most predicted translation repression targets did not show appreciable changes in transcript abundance in the absence of sRNAs. Additionally, of three candidate targets examined at the protein level, only one showed a moderate variation in polypeptide amount in the mutant strain. Our results emphasize the difficulty in identifying genuine miRNA targets in Chlamydomonas and suggest that miRNAs, under standard laboratory conditions, might have mainly a modulatory role in endogenous gene regulation in this alga.