Small RNA-Dependent Gene Silencing in the Green Alga Chlamydomonas reinhardtii: Functions and Mechanisms

Small RNAs (sRNAs), ~20-30 nucleotides in length, are non-coding RNAs that play essential roles in the regulation of gene expression in eukaryotes. They lead to inactivation of cognate sequences at the post-transcriptional level via a variety of mechanisms involved in translation inhibition and/or RNA degradation. In the Chlorophyta Chlamydomonas reinhardtii, however, the molecular machinery responsible for sRNA-mediated translational repression remains unclear. To address the mechanisms of translation inhibition by sRNA, we have isolated an RNAi defective mutant (Mut26), which contains a deletion of the gene encoding the homolog of CCR4 in Chlamydomonas. We investigated the expression of both an exogenous siRNA target and endogenous miRNA target. Additionally, the pattern of poly(A) tailing in diagnostic mRNAs was examined with the G/I tailing assay and CCR4 partner proteins were identified through affinity purification. Our overall results are consistent with the role of CCR4 in sRNA-dependent translational repression without target mRNA degradation in Chlamydomonas. The biological function(s) of miRNAs in responses to nutrient deprivation in Chlamydomonas reinhardtii were also explored. Transcriptome analysis using cells grown under various trophic conditions revealed that several miRNAs were differentially expressed, but their predicted targets showed no changes in transcript abundance. Collective evidence suggests that miRNAs may not play an essential role in endogenous gene regulation in Chlamydomonas