The long-term objective of the proposed research will be to gain an understanding of RNA structure and function with an emphasis on RNA catalyzed reactions. Diseases due to RNA splicing defects, such as certain forms of thalassemia, are due to mutations which affect accuracy in a reaction for which RNA may play an important catalytic role. The system that will be studied is derived from the self-splicing IVS encoded in the Tetrahymena macronuclear pre-rRNA gene. Altered forms of the IVS will be studied as to the effects on splicing and reverse splicing activity, stability, and a novel form of the IVS catalyzed RNA polymerase activity. All group I IVSs have the potential to form a conserved secondary structure (involving about 100 of the 413 nucleotides in the Tetrahymena IVS). It is hypothesized that this conserved structure forms the """"""""core"""""""" three dimensional structure which is the catalytic center of the IVS. Most studies involving mutagenesis have been directed at testing specific features of the core structure. Random mutagenesis has not been fully exploited in this system as a means of identifying critical sequences and structures. Random mutations, both single-base changes and linker insertions, will be made to examine the role of both core and non-core structures. Mutations in the core will, most likely, affect catalytic activity directly whereas mutations outside of the core may be expected to affect overall stability or flexibility of the IVS. The linker library will be used to generate linker scanner mutants and nested deletions of specific sequences and structures. Although each step in splicing appears to be reversible, the total reverse reaction, IVS integration, has not been demonstrated. The forward direction may be driven by circularization, which removes one of the products of splicing. An in vitro system which will detect reverse splicing will be developed using mutants which do not circularize. Results from these experiments would be of significance to the question of whether self-splicing RNA could act as mobile genetic elements. A variation of the nucleotidyl transferase reaction, catalyzed by a portion of the IVS, has been found in which a primer is elongated in the 5' to 3' direction by the successive addition of mononucleotides. The potential for this reaction to be template directed will be studied.
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