Flagellum ontogeny in trypanosomes studied via an inherited and regulated RNA interference system

The African trypanosome, Trypanosoma brucei possesses a large and unique in traflagellar structure called the paraflagellar rod (PFR). The PFR is compo sed of 2 major proteins, PFRA and PFRC, We have generated an inducible muta nt trypanosome cell line (snl-2) that expresses linked inverted copies of a PFRA gene, capable of forming a PFRA double-stranded (ds) RNA, When expres sion of this dsRNA was induced, new PFRA RNA and PFRA protein quickly disap peared and PFR construction was affected, resulting in cell paralysis. This inducible RNA interference (RNAi) effect was fast-acting, heritable and re versible. It allowed us to demonstrate that PFR proteins are able to enter both mature and growing flagella but appear to concentrate differentially i n new flagella because of the construction process. The PFR is constructed by a polar assembly process at the distal end of the flagellum resulting in a stable cytoskeletal structure with low turn-over. The inducible RNAi app roach will have widespread applicability in studies of gene function and ce llular processes in parasites.