kRNA editing is a novel type of RNA processing found in kinetoplastids which alters the sequence of many mitochondrial RNAs by posttranscriptional uridine insertion and deletion to form functional mRNAs. Editing is so extensive in Trypanosoma brucei over half of the nucleotides in many mature mRNAs result from posttranscriptional uridine insertion. The sequence information for editing is supplied by small RNAs termed guide RNAs (gRNAs), which are complementary to edited mRNA sequence. However, aside from gRNAs, the trans-acting factors involved in kRNA editing are largely unknown. Recently, an in vitro assay for deletion-type kRNA editing in T. brucei mitochondrial extracts was developed and, using this assay, deletion editing activity has been localized to 20S particles in glycerol gradients. This proposal is designed to affinity purify the 20S """"""""editosome"""""""", identify editosome- specific proteins, isolate their genes, and assess protein function and interaction with editing-related complexes. Editosomes will be purified using a three-step affinity procedure based on selection of tag sequences on exogenously added gRNA and mRNA known to interact with the editosome in vitro. Following purification, the molar ratio of component proteins and their general characteristics, such as RNA binding and phosphorylation state, will be investigated. These experiments will both provide an analysis of editosome composition and help to identify the most important and promising candidates for protein purification. One or two selected editosome proteins will be isolated, their genes cloned, sequenced, and expressed, and antibodies produced. Genomic organization will be ascertained and transcript size and abundance throughout the T. brucei life cycle will be determined. Predicted amino acid sequences will be analyzed in order to provide clues to protein function. More direct assessment of protein function will be made using antibodies to block, immunoprecipitate, or deplete mitochondrial extracts of editing activity, putative editing-related enzyme activities, and RNA binding capacity. Immunoprecipitation of mitochondrial complexes containing the antigen will permit characterization of their protein and RNA content and possibly provide a convenient means for immunopurification of additional editing- related proteins. These experiments will identify components of the machinery which catalyzes kRNA editing and will increase our understanding of the mechanism and regulation of this novel genetic regulatory process. In addition, they will potentially provide molecular tools for investigation of related processes in other organisms.
