Characterisation of the ATP binding motif of PRP2

• Main Author: Flinn, Elizabeth M.
• Published: University of Edinburgh 1994
• Subjects: 572.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.650975

Four dominant negative mutants of PRP2 were produced by site-directed mutagenesis of the conserved ATP-binding motif (Motif A) of PRP2. These mutants, when overproduced in a wild-type yeast strain caused a growth inhibition due to inhibition of splicing, leading to an accumulation of pre-mRNA. <I>In vitro</I> analysis of cell-free splicing extracts showed that splicing was inhibited in the presence of the dominant negative proteins. The splicing inhibition could be alleviated if enough wild-type PRP2 was present in the extracts. The dominant negative mutants caused accumulation of the fully assembled but inactive spliceosome. Immunoprecipitation with anti-PRP2 antibodies showed that one mutant protein (PRP2<SUP>GKN</SUP>) was stably associated with the spliceosome. In contrast, another mutant protein (PRP2<SUP>AKT</SUP>), appeared not to be associated with the spliceosome, suggesting that it was sequestering some other essential splicing factor outside the splicing complex. Using an affinity column, two of the dominant negative proteins have been purified. Assays have shown that neither protein can bind poly U RNA and that both show negligible ATPase activity. Both purified proteins can inhibit the activity of a wild-type splicing extract and prevent the formation of the active spliceosome.