Differential enzymatic accessibilities of the 5' and 3' splice sites of beta-globin pre-messenger RNA in splicing competent HeLa cell nuclear extract

Inhibition of oligonucleotide-directed cleavage of pre-mRNA using exogenously added E. coli RNase H has been utilized as a probe for mRNA-protein interaction. We now show that such an RNase H-like activity is present in splicing competent Hela cell nuclear extract. Using this extract and in vitro transcribed beta-globin pre-mRNA, we have demonstrated that synthetic oligonucleotides, complementary to the splice site sequences, direct preferential cleavage of the 5' splice site. Thus, these experiments using complementary oligonucleotide-directed, endogenous RNase H-like cleavage of pre-mRNA, suggest a useful probe for studying the mRNA-protein complex in vitro.