We describe here a forward genetic screen to investigate the biogenesis, mode of action and biological function of miRNA-mediated RNA silencing in the model algal species Chlamydomonas reinhardtii. Amongst the mutants from this screen there were three at Dicer-like 3 that failed to produce both miRNAs and siRNAs and others affecting diverse post-biogenesis stages of miRNA-mediated silencing. The DCL3-dependent siRNAs fell into several classes including transposon- and repeat7 derived siRNAs as in higher plants. The DCL3-dependent miRNAs differ from those of higher plants, however, in that many of them are derived from mRNAs or from the introns of pre-mRNAs. Transcriptome analysis of the wild type and dcl3 mutant strains revealed a further difference from higher plants in that the sRNAs are rarely negative switches of mRNA accumulation. The few transcripts that were more abundant in dcl3 mutant strains than in wild type cells were not due to sRNA-targeted RNA degradation but to direct DCL3 cleavage of miRNA and siRNA precursor structures embedded in the untranslated (and translated) regions of the mRNAs. This analysis reveals that the miRNA pathway in C. reinhardtii differs from that of higher plants and resembles that of animals in terms of miRNA precursor structure, domain structure of DCL and in that the target mRNAs persist in the presence of miRNAs. Altogether, our findings inform the understanding of the evolution and function of miRNA-mediated RNA silencing in eukaryotes.
- RNA silencing