U-insertion/deletion RNA editing is a unique post-transcriptional RNA modification process occurring in the mitochondria of trypanosomatid parasites. Editing modifies the sequences of 12 of 16 mitochondrial mRNAs transcribed from mitochondrial DNA "cryptogenes" by insertion and deletion of uridines at specific sites to render them translatable. Our discovery of a new class of short RNAs (guide RAs or gRNAs) that contain the sequence information for editing by base pairing in trans brought this type of RNA modification into line with the central dogma. The hypothetical editing model we proposed based on the mediation of gRNAs involved multiple 3'-5'cycles of cleavage, U-addition or U-deletion and ligation. The proteins responsible for the enzymatic activities we proposed for this model were identified as components of a 1 md multiprotein complex, the RNA Editing Core Complex or RECC. Several additional multiprotein complexes which may be involved in RNA regulation interact with the RECC via RNA linkers. Editing occurs in all members of the Kinetoplastid lineage, which includes the trypanosomatid parasites, the causal agents of several human diseases. The understanding of editing cannot proceed without knowledge of the molecular architecture of the editing complexes and their interactions with substrate RNAs. We propose to perform a high resolution structure- function analysis of the main component of the editing reaction - the 1 md RECC. We will attempt to confirm and extend our hypothetical editing reaction model by determining the 3D structure of these particles at an atomic resolution and identifying the protein components and their protein-protein and protein-RNA interactions in different functional states of the editing reaction The molecular structure information will be interpreted in terms of the detailed mechanism of this pathway and this knowledge will provide a basis for future investigations of the holoenzyme and the possible interactions of editing with mitochondrial RNA regulation and protein translation. This is important since the trypanosomatids have become important eukaryotic model systems for studying many basic biological phenomena in addition to RNA editing, such as protein expression, trans-splicing surface antigen variation and flagellar motility. Since it now appears that mitochondrial RNA editing may interact with translation and RNA regulation, an understanding of editing will assist in the development of trypanosomatids as model biological systems. In addition, U-insertion/deletion editing serves as a paradigm for multiple other post-transcriptional RNA modification phenomena such as fold-back guided A to I editing in human brain, sno-RNA guided methylation and pseudouridylation of ribosomal RNAs and even Si RNA-guided cleavage of mRNAs in RNAi, and the information obtained in this project may prove relevant for understanding these other biologically important phenomena. And last but not least, the editing pathway is essential for viability and may provide a potential target for development of interventions against the diseases caused by these parasites.
This project proposes to obtain a functional high resolution 3D structure for the core multiprotein complex involved in trypanosome mitochondrial uridine insertion/deletion RNA editing in which U's are inserted and deleted within coding regions of mRNAs. Binding and processing of substrate RNAs during the in vitro editing reaction will also be analyzed structurally. The biomedical significance is that these parasites are the causal agents of a number of important human diseases including Chagas Disease, Leishmaniasis and African Sleeping Sickness, and that the editing pathway is essential in all stages of the parasite life cycles and not present in the human host, thereby making it a potential target for therapeutic intervention against these parasites.
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