Journal of Virology · 1983 · 133 citations · 25 references
Life SciencesMicrobiologyMolecular BiologyMolecular VirologyRna ProcessingVirologyRibosomal Rna ProcessingRna CleavageViral Rna BiologyAgarose Gel ElectrophoresisCell-free Culture SystemsEndonucleolytic Cleavage Of Ribosomal RnaEndonucleolytic Rna CleavageMolecular FragmentsPre-ribosomal Rna CleavageSmall Subunit Ribosomal Rna ProcessingViral Rna Cleavage
2‑5A activates a latent endoribonuclease that cleaves rRNA into characteristic fragments, a process previously inferred only from electrophoretic comigration. The study proposes using rRNA cleavage patterns as an alternative assay to detect 2‑5A system activity. Northern blotting identified 18S/28S origins of rRNA cleavage fragments, with identical 3′ termini in both cell‑free and infected cells, demonstrating that cleavage patterns reliably indicate prior 2‑5A‑dependent RNase activity and that the assay is convenient and unambiguous. © 1981, Nucleic Acids Research 9:1571‑1581.
In cell-free systems, 2-5A [ppp(A2'p)nA, n = 2 to greater than or equal to 4] activates a latent endoribonuclease, the 2-5A-dependent RNase, which cleaves rRNA in intact ribosomes into discrete and characteristic products (D. H. Wreschner et al., Nucleic Acids Res. 9:1571-1581, 1981). Here we present Northern blots which have identified the 18S or 28S origins of the cleaved products from rRNA. In addition, identical 3' termini were observed for fragments of 18S rRNA from a HeLa cell-free system incubated with 2-5A and from interferon-treated, encephalomyocarditis virus-infected HeLa cells. The previous assumption of identity of such fragments was based only on comigration on electrophoresis in agarose gels. We conclude that appropriate patterns of cleavage found in RNA isolated from intact cells are an indicator of prior 2-5A-dependent RNase activity. The assay of rRNA cleavage is relatively convenient and unambiguous. Accordingly, in the search for situations in which the 2-5A system may be active, it provides a useful alternative to the direct assay of 2-5A.
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