Small Interfering RNA–Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide inChlamydomonas reinhardtii

Small RNAs (sRNAs; ∼20 to 30 nucleotides in length) play important roles in gene regulation as well as in defense responses against transposons and viruses in eukaryotes. Their biogenesis and modes of action have attracted great attention in recent years. However, many aspects of sRNA function, such as the mechanism(s) of translation repression at postinitiation steps, remain poorly characterized. In the unicellular green alga Chlamydomonas reinhardtii, sRNAs derived from genome-integrated inverted repeat transgenes, perfectly complementary to the 3' untranslated region of a target transcript, can inhibit protein synthesis without or with only minimal mRNA destabilization. Here, we report that the sRNA-repressed transcripts are not altered in their polyadenylation status and they remain associated with polyribosomes, indicating inhibition at a postinitiation step of translation. Interestingly, ribosomes associated with sRNA-repressed transcripts show reduced sensitivity to translation inhibition by some 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 C. reinhardtii may involve alterations to the function/structural conformation of translating ribosomes. Additionally, sRNA-mediated translation inhibition is now known to occur in a number of phylogenetically diverse eukaryotes, suggesting that this mechanism may have been a feature of an ancestral RNA interference machinery.