The ribosomal RNA of Tetrahymena thermophila is produced as a precursor containing an intervening sequence (intron) which is able to catalyze its own excision and to join its flanking sequences (exons). Given the increasingly central role RNA catalysis may play in cell function, it is essential in the long-term to determine the structure of this catalytic RNA and the function of each of its domains. We plan to ask how the intron recognizes the junction between itself and the downstream exon which is known as the 3' splice-site (3'SS). Study of the 3'SS is particularly interesting because the catalytic RNA has to ligate the exons by transesterification while at the same time preventing abortive hydrolysis of the 3'SS. Most other catalytic RNAs simply have to cleave a substrate. It is essential to know both the minimal features required to determine a 3'SS as well as the features required to ensure that only the desired 3'SS is selected. The first aspect will be studied by analyzing mutations deliberately made in the region of the 3'SS. The second will be studied by analyzing the effect of providing various competing 3' splice-sites upstream of the normal 3'SS. A recently discovered cryptic 3'SS and a duplication of the normal 3'SS will be used. By using oligonucleotide directed mutagenesis to make mutations in the normal or the competing 3'SS the features which contribute to 3'SS selection will be identified. Splicing assays will be performed in vitro using synthetically made RNA precursors. A region of the intron that interacts with the upstream splice site has been clearly identified but no such region has been identified for the 3'SS. We will search for it by screening for mutations which only inhibit 3' splicing and by screening for suppressors of mutations which map at the 3'SS. Suppressor mutations which map elsewhere in the intron and restore splicing are likely to lie in regions which interact with the 3'SS. Forward mutations and suppressors can be selected because we have inserted the intron into a gene of E. coli in such a way that the gene is only expressed if the intron splices itself out. The competitive splice-site analysis will also be aided by this in vivo splicing assay.
