RNA editing in Trypanosoma brucei is a process that inserts / deletes uridylate residues (U) from mitochondrial pre-mRNA molecules. This phenomenon produces mature mRNAs by creating open reading frames, correcting frame shifts and creating signals for translational initiation and termination. The placement of Us is guided by a small RNA molecule (the guide (g) - RNA) which is complementary to portions of the mature mRNA. While the gRNA is known the carry the information for the editing process, little is known about how the gRNAs are used to direct the editing process. All of the gRNAs identified to date have defined 5' anchor sequences, guiding sequences and a non-encoded 3' uridine tail. The overall objective of this proposal is to understand how the different gRNA elements contribute to the editing process, how they interact with the pre-edited mRNA during editing and ultimately how the gRNA is utilized to direct editing. This objective will be approached using three avenues of research: 1) an analysis of the secondary structure interactions between gRNA and mRNA during the editing process. The extent of the gRNAs interactions with the mRNA will be determined by combining photoaffinity cross linking with structure probing techniques. Defining the structures of the gRNA and the mRNA as they interact during the editing process will help lead to a molecular understanding for the role of the gRNAs in the editing process. 2) An analysis of the tertiary structure of RNAs and proteins in the editing complex. The cross linking patterns of photo-agents attached at homologous sites in different gRNAs and mRNAs will be analyzed to determine if these RNAs contain a common, core tertiary structure. Using site-specific cross linking techniques, the positions of editing proteins in relation to specific regions of the gRNA and mRNA will be analyzed. 3) A determination of the roles of sequence and structure in the kinetics of gRNA/mRNA association. The contribution both sequence and structure make in the overall kinetics of RNA interaction will be analyzed using native and temperature gradient gel electrophoresis and surface plasmon resonance technology. The binding of the gRNA to the mRNA is the fundamental step in RNA editing. An understanding of the nature and relative importance of the elements, which confer specificity on this interaction, is critical to our understanding of the editing process. This research will allow us to begin to understand the structural function relationship of RNAs and protein within the editing complex and greatly enhance our understanding of the mechanism involved in the transfer of information from one RNA molecule to another. Members of the kinetoplastida are the causative agents of African sleeping sickness, Chagas disease and leishmaniasis. Understanding kRNA editing, which is unique to these organisms, will contribute to the fundamental knowledge of the parasite and may lead to the development of new strategies for disease intervention and control.
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