Mitochondrial translation apparatus of trypanosomatids still is in the early investigation stage. This system operates with templates some of which need to be extensively edited prior to translation, one can anticipate that this system will be quite different from that in other systems including mammals. Revealing and understanding of these differences is not only interesting but can also lead to their therapeutic exploitation for better treatment of the diseases caused by trypanosomatids. The recent analysis of mitochondrial ribosomes in a model organism, Leishmania tarentolae, has also demonstrated the presense of unique heterodimeric 45S SSU* complexes representing an association of the ribosomal small subunit with a protein complex of a yet poorly understood nature. Such a complex is unprecedented and its function is unknown. It is possible that this complex is involved in recognition of competent templates for subsequent translation, although there are no data to substantiate this hypothesis. The goal of this proposal is to investigate the presence of mitochondrial ribosomes and the related complexes in in Trypanosoma brucei and to employ gene knock-down by RNA interference in order to probe into potential functions of the 45S SSU* complexes. This organism is chosen because it is particularly amenable to the RNAi approach.
Specific aim 1 : Investigation of mitochondrial ribosomal RNP complexes in Trypanosoma brucei. The existence of mitochondrial ribosomal ribonucleoprotein (RNP) complexes, primarily 45S SSU* complexes, will be investigated in T. brucei. Detergent lysates of purified mitochondria will be analyzed by velocity sedimentation in sucrose gradients. Ribosomal complexes will be detected by investigating sedimentation profiles of the ribosomal small subunit (9S) and large subunit (12S) ribosomal RNAs. The identity of the complexes will be further investigated by probing gradient fractions with antibodies that will be produced against conserved small subunit and large subunit ribosomal proteins and presumtive 45S SSU*-specific proteins. A mass-spectroscopy analysis of the purified complexes 45S SSU* will be performed to ascertain their protein composition in this species.
Specific aim 2 : Functional evaluation of the 45S SSU* complexes by RNA interference. The function of the 45S SSU* complex will be probed into by investigating the impact of down-regulating several of its protein components on the integrity of the complexes using RNAi. The targets will include several 45S-specific proteins, mainly the PPR proteins found in this complex. The target mRNA and protein levels will be investigated daily over the period of 7-14 days since the induction of RNAi to estimate the efficacy of the knock-downs, in addition to growth kinetics. At a selected time point(s), the abundance (relative unindiced cell) of the 45S SSU* complexes, as well as monosomes and subunits, will be investigated. The effect of the RNAi on the de novo synthesis of the mitochondrial translation products Cyb and COI will be investigated by studying relative levels of 35S-incorporation into the respective polypeptides. The effect on the stability of the polypeptides and their assembly into the respiratory complexes will be studied by Blue Native gel electrophoresis.
Trypanosoma brucei belongs to the group of parasitic protists which cause the diseases affecting millions of people worldwide. The current treatments of these diseases are not satisfactory. The project is aimed at investigation of T. brucei mitochondrial ribosomes and other components of the protein synthesis system that plays an indispensable role during the parasite's life cycle. This system has a potential as a target for chemotherapy.
