Role of RNase H in hybrid-arrested translation by antisense oligonucleotides.
The mechanism of hybrid-arrested translation by antisense oligodeoxynucleotides has been investigated with the rabbit reticulocyte lysate system. The oligonucleotides studied were directed against different regions of mouse alpha- or beta-globin mRNAs. Freshly prepared reticulocyte lysates were found to contain 1-2% of the level of RNase H in nucleated cells. This level of activity was sufficient to cleave nearly 100% of the targeted mRNA at the site of hybridization with a complementary oligodeoxynucleotide in 1 hr under conditions of active translation. Using poly(rA).oligo(dT) as a competitive inhibitor of the enzyme, hybrid arrest by oligodeoxynucleotides complementary to the sequence spanning the initiation codon or to a sequence in the coding region was found to be due entirely to cleavage of mRNA by RNase H. Hybridization of oligodeoxynucleotides adjacent to the cap site of beta-globin mRNA, but not the alpha-globin mRNA, also inhibited protein synthesis directly. Even in this case, however, cleavage of the mRNA by RNase H was the predominant pathway of inhibition.
Walder
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Department of Biochemistry, University of Iowa, Iowa City 52242.
Walder
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JA
D000818:Animals; D003062:Codon; D004247:DNA / Q000235:genetics; D004722:Endoribonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005260:Female; D005914:Globins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D009841:Oligonucleotides / Q000235:genetics / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense; D011061:Poly A / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D016914:Ribonuclease H
D003062:Codon; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011061:Poly A; C018368:poly(rA).oligo(dT); D005914:Globins; D004247:DNA; D004722:Endoribonucleases; D016914:Ribonuclease H