? The goal of this FIRCA proposal is to assess the roles of three guide RNA binding proteins, gBP21, gBP25, and TbRGG1. These are essential for editing but are not integral components of the complex that catalyzes editing (editosome) and thus appear to be accessory factors. The project will generate transgenic trypanosomes with regulatable knockdown of expression of one or simultaneously two genes and regulatable knock-in of TAP-tagged normal genes and those that are mutated to disable protein function. Resultant transgenics will be generally characterized initially. The effects of regulated gene inactivation or expression of disabled genes in the transgenics on steps in the editing process will be determined in vivo and in vitro by analysis of edited products and the component activities of editing utilizing methods developed in the Stuart lab. The effects on editosome structure will be examined by density gradient sedimentation combined with Western analyses including the use of monoclonal antibodies made in the Stuart lab. Associations among the accessory proteins will be examined by analysis of TAP-tagged purified proteins including the use of tandem mass spectrometry, which is routine in the Stuart lab. The integrity and composition of respiratory complexes, some components of which are encoded in edited mRNAs, will also be characterized. The roles of the gRNA binding proteins may include stage-specific regulation of editing, processing of multigenic transcripts, or transcript turnover. This FIRCA project complements the parent grant that is focused on identification of the stable components catalytic complex, their functions, and the editing cycle. Each laboratory has complementary skills and materials thus providing for project synergy. The Lukes lab will benefit from expertise and materials that will be transferred form the Stuart lab including monoclonal antibodies and cell lines with mutated editosome genes. The Stuart lab will benefit from the new RNAi cell lines that may be arrested at specific steps in editing and the new knowledge generated from them. ? The research will be done as an extension of an NIH grant. ? The research will be done in collaboration with Julius Lukes, Ph.D. at the Institute of Parasitology in the Czech Republic. ? ?
