The main focus of this renewal application is the role of exon sequences in alternative splicing. The proposed experiments following from observations made during the previous funding period emphasizing the importance of the contribution of exon sequences in the retention or exclusion of alternative exons. While the previous experiments relied on mutation of individual elements, the new approach is modified on SELEX. Basically, random 20mers will be introduced into constructs in which the internal exon is excluded from the product and selected for exon sequences which now result in inclusion. The proposed experiments will test quantitatively the effects of length and sequence context. In a similar approach, constructs will be selected in which skipping of the internal exon will be selected from a starting construct which is included. These experiments are argued to be of particular interest since negative exon sequence elements have been found rarely. Finally, trans-acting factors which bind to these putative enhancer (or silencer) elements will be identified using competitor RNAs as probes in gel shift experiments and crosslinking and/or immunoprecipitations with antibodies to known hnRNP and SR proteins. If novel factors are identified, their purification will be undertaken.
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