Structure-function in Xenopus snRNPs

Abstract

For this analysis an in vivo assay in Xenopus oocytes is being used which is based on the principle that, using site directed hydrolysis by RNAse H, a specific snRNA population can be destroyed by nuclear injection of an oligodeoxynucleotide, complementary to a part of an snRNA sequence (Pan and Prives, 1988). This will inhibit the function of the targeted snRNA. The snRNA population can be restored by coinjection of a plasmid containing the gene for the targeted snRNA (Hamm et al., 1989). The function is assayed by a second injection with a 32p-labeled pre-mRNA and subsequent test of splicing. By coinjection of genes coding for mutant snRNAs one can test whether the transcripts of these genes can complement splicing in vivo. Simultaneously the assembly of of the newly formed snRNP particles into spliceosomes can be assayed using native polyacrylamide gels.