Short tandem target mimics inhibit Chlamydomonas reinhardtii microRNAs

Abstract Short tandem target mimic (STTM) RNAs contain two non-cleavable microRNA (miRNA) binding sites linked by a spacer and can be used to effectively block the functions of specific miRNAs in animals and plants. However, the application of STTMs in unicellular organisms has not been reported. In this study, we tested different STTMs targeting miRNAs of the microalga Chlamydomonas reinhardtii to verify the feasibility of this approach in a unicellular organism. Three previously identified C. reinhardtii miRNAs (miR906-5p, miR1166.1, and miR1150.3) were used as targets and transgenic algae containing STTMs were constructed. All three STTM constructs induced significantly lower miRNA levels, from 84% lower levels of miR1150.3 to 73% lower levels of miR1166.1 compared with non-transgenic controls. Surprisingly, this effect on miRNA levels was non-specific, independent of its target miRNA sequence and each STTM suppressed all miRNAs tested. This is the first time that the STTM technique has been tested in a lower unicellular organism. The non-specific inhibition of miRNA expression by the STTMs revealed the complexity of miRNA functions in diverse eukaryotic organisms.