RNA interference mediated by translation repression and the role of raptor, the regulatory associated protein of TOR (target of rapamycin)

Tomohito Yamasaki, Xinrong Ma, Eun-Jeong Kim, Fadia Ibrahim, and Heriberto Cerutti

School of Biological Science and Center for Plant Science Innovation, University of Nebraska, Lincoln, NE 68588, USA

Small RNAs (~20-30 nucleotides in length) silence gene expression in a broad range of eukaryotes. These noncoding RNAs usually trigger post-transcriptional degradation and/or translational repression of complementary target transcripts. In Chlamydomonas reinhardtii, RNA interference (RNAi) can be induced by inverted repeat (IR) transgenes that produce double-stranded RNA homologous to a gene of interest. Intriguingly, strain 44s, containing an IR transgene targeting MAA7 (encoding tryptophan synthase beta subunit), showed significantly reduced levels of the TSB protein without any marked change in the MAA7 mRNA amount. Moreover, the MAA7 transcript was found associated with polysomal fractions in sucrose density gradients. These results suggested that small interfering RNAs (siRNAs) perfectly complementary to a target mRNA can inhibit translation at a post-initiation stage in Chlamydomonas. To gain insight into this mechanism, we carried out random insertional mutagenesis using 44s as the host strain and isolated several RNAi-defective mutants. Mut-19 displayed a partial deletion of the raptor gene. Transformation of this mutant with epitope-tagged raptor recovered RNAi-mediated translation repression. By affinity purification and MS analyses, we identified a homolog of translation elongation factor 3 and the TOR kinase as interacting partners of raptor. However, direct association with known components of RISC was not detected. The target of rapamycin (TOR) kinase interacts with raptor to form a protein complex, which plays a central role in the regulation of cell growth in response to environmental cues. Our findings suggest that TOR and Raptor are also involved in siRNA-mediated translational repression in Chlamydomonas.

e-mail address of presenting author: yamasaki.tomohito@gmail.com