The focus of this renewal application is the structure and function of aI2, a self-splicing intron from yeast mitochondria. In previous work it was shown that the ORF 1) is a maturase, 2) is a site- specific mobile element, and 3) contains homology to reverse transcriptases (RT), DNA endonucleases (Zn), and retroviral proteases (P). There are three major specific aims. 1) The aI2 intron is expressed as a 96kD precursor, and processed to p62 which contains RT, X and Zn. The applicant will determine if p62 also contains P and Z, a domain found in some non-LTR retroelements, and whether these sequences are important for splicing and/or mobility. 2) To determine the role of the maturase in splicing, two approaches are proposed. First, the PI will test the specific hypothesis that p62 promotes proper folding of the intron by looking for RNA-protein interactions using a variety of standard techniques. Associations with other proteins will also be assayed using the knowledge that p62 sediments as several RNPs. These biochemical approaches will be supplemented with information obtained from the creation of aI mutants and suppressors. Second, he will attempt to develop a p62-dependent splicing system, based on the fact that aI does not self-splice at low salt in vitro. Both forward and reverse splicing assays are described. 3) A working model for group II intron mobility will be tested which involves reverse transcription of the aI2- containing pre-mRNA and site-specific insertion into recipient, intronless genes. In addition to RT, Zn is essential, perhaps by providing the primer for first-strand cDNA synthesis. The presumptive case for an RNA intermediate will be definitively tested a la Boeke and Fink. The relationship of splicing to mobility will be investigated by the analysis of mutants, including the possibility that the excised intron is involved. The role of exon sequences in recipient and donor will be distinguished and the in vivo mobility requirements will be compared to those for a new in vitro endonuclease assay (to be carried by the Lambowitz laboratory). Finally, the applicant will investigate unusual mobility events which may involve an RT-independent pathway and a reverse-splicing-dependent pathway.
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