GENETIC AND MOLECULAR STUDIES OF RNA SPLICING

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The long-term goals of this research are to 1) develop new insights to the structure of group II introns of yeast mtDNA and roles of those structures in splicing reactions in vivo and in vitro; 2) study the function of a maturase protein encoded by a group IIA intron in splicing; 3) develop and test models for two genetic phenomena dependent on the group IIA introns; and 4) purify and characterize mutant and wild-type proteins encoded by group II introns. The validity of predicted tertiary structures will be tested in vivo by transforming relevant mutant alleles into mtDNA; the in vivo phenotypes will be determined and second-site revertants isolated and characterized. We have discovered a crucial tertiary interaction between domain 5 and domain 1 of group II introns that is necessary for reactions at both splice junctions. Further insight to the functional importance of specific sequences and structures within domain 5 will be obtained by screening randomized domain 5-containing RNAs for retained function using a trans-splicing assay. Mutants of domain 5 will be transformed into mitochondria and second-site suppressors characterized in order to define regions of the intron with which it interacts. The reading frame of the group IIA intron resembles reverse transcriptase and also contains a zinc-finger like domain; nearly all group II introns with reading frames have those features and additionally other highly conserved segments not resembling a known protein are present. We will mutate each of those regions, transform the mutations into mitochondria and determine the effect on splicing. Reading frame mutants will be tested for their effect on two genetic phenomena that appear dependent on that intron, namely, reversion of splicing defective mutants by intron excision (RIE) and group II intron mobility. Biochemical experiments will be carried out to test predictions of models for those processes. And finally, wild-type and mutant forms of proteins encoded by group II introns will be purified from mitochondrial extracts and characterized; in vitro assays for splicing, nucleic acid splicing and/or reverse transcriptase will be tested.