Small interfering RNA-mediated translation repression alters ribosome sensitivity to inhibition by cycloheximide in Chlamydomonas reinhardtii

RNA interference (RNAi) is an evolutionarily conserved gene silencing mechanism in eukaryotes, with regulatory roles in a variety of biological processes, including cell cycle, cell differentiation, physiological and metabolic pathways, and stress responses. RNAi can function by transcriptional silencing, mRNA target cleavage, translation repression and/or DNA elimination. In this study, we used the unicellular green alga Chlamydomonas reinhardtii as a model system to study RNAi-mediated translation repression. We demonstrated that small RNAs (sRNAs) generated from exogenously introduced inverted repeat transgenes, with perfect complementarity to the 3\u27UTR of a target transcript, can inhibit protein synthesis, without or with only minimal mRNA destabilization. In addition, there are no changes in the polyadenylation status of sRNA-repressed transcripts. Moreover, the translationally repressed mRNAs remain associated with polyribosomes, suggesting that sRNA-mediated silencing occurs at a post-initiation step of translation. Intriguingly, we consistently observed reduced sensitivity of the ribosomes associated with these repressed transcripts to inhibition by antibiotics such as cycloheximide, both in ribosome run-off assays and in in vivo experiments. Our results suggest that sRNA-mediated repression of protein synthesis in Chlamydomonas may involve alterations to the function/structural conformation of translating ribosomes. Additionally, since sRNA-mediated translation inhibition is now known to occur in a number of phylogenetically diverse eukaryotes, this mechanism may have been a feature of an ancestral RNAi machinery