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.
Siegall
C B
CB
Department of Biochemistry, George Washington University School of Medicine and Health Sciences, Washington D.C. 20037.
Kumar
A
A
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005914:Globins / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D016914:Ribonuclease H; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D005914:Globins; D004722:Endoribonucleases; D016914:Ribonuclease H