Ribozymes correctly cleave a model substrate and endogenous RNA in vivo.
The alpha-sarcin domain of 28 S RNA in Xenopus oocytes is attacked by several catalytic toxins (e.g. alpha-sarcin and ricin) that abolish protein synthesis. We synthesized 6 ribozymes targeted to the alpha-sarcin domain and to an oligoribonucleotide (34-mer) that mimics this domain. Sarcin ribozyme 5 (SR5) efficiently cleaved after the CUC site in the synthetic 34-mer in vitro at 50 degrees C. SR5 also cut the same site when both substrate and ribozyme were coinjected or injected separately into oocytes at 18 degrees C. Correct cleavage in vivo was shown by isolating and sequencing the large cleavage fragment. The cleavage reaction appeared to function equally well in the oocyte nucleus and cytoplasm. SR5 also correctly cleaved endogenous 28 S RNA in oocytes, although cutting was much less efficient than with alpha-sarcin. We therefore demonstrated that a ribozyme specifically cuts both a model substrate and a cellular RNA in vivo. Earlier work showed that certain injected deoxyoligonucleotides complementary to the alpha-sarcin region abolish protein synthesis. Oocyte protein synthesis was also abolished by an SR5 containing a single G----U substitution that inactivates RNA catalysis, indicating that SR5's translational suppression was perhaps due to antisense function rather than ribozyme cleavage.
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
S K
SK
Saxena
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EJ
Ackerman
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D003513:Cycloheximide / Q000494:pharmacology; D004722:Endoribonucleases; D005260:Female; D005656:Fungal Proteins / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012339:RNA, Ribosomal, 28S / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014981:Xenopus
D005656:Fungal Proteins; D015345:Oligonucleotide Probes; D011500:Protein Synthesis Inhibitors; D016337:RNA, Catalytic; D012339:RNA, Ribosomal, 28S; C014801:alpha-sarcin; D003513:Cycloheximide; D008715:Methionine; D004722:Endoribonucleases