FINE MAPPING OF 28S RIBOSOMAL-RNA SITES SPECIFICALLY CLEAVED IN CELLSUNDERGOING APOPTOSIS

Bona fide apoptosis in rat and human leukemia cells, rat thymocytes, a nd bovine endothelial cells was accompanied by limited and specific cl eavage of polysome-associated and monosome-associated 28S rRNA, with 1 8S rRNA being spared. Specific 28S rRNA cleavage was observed in all i nstances of apoptotic death accompanied by internucleosomal DNA fragme ntation, with cleavage of 28S rRNA and of DNA being linked temporally. This indicates that 288 rRNA fragmentation may be as general a featur e of apoptosis as internucleosomal DNA fragmentation and that concerte d specific cleavage of intra- and extranuclear polynucleotides occurs in apoptosis. Apoptosis-associated cleavage sites were mapped to the 2 88 rRNA divergent domains D2, D6 (endothelial cells), and D8. The D2 c uts occurred in hairpin loop junctions considered to be buried in the intact ribosome, suggesting that this rRNA region becomes a target for RNase attack in apoptotic cells. D8 was cleaved in two exposed UU(U) sequences in bulge loops. Treatment with agents causing necrotic cell death or aging of cell lysates failed to produce any detectable limite d D2 cleavage but did produce a more generalized cleavage in the D8 re gion. Of potential functional interest was the finding that the primar y cuts in D2 exactly Banked a 0.3-kb hypervariable subdomain (D2c), al lowing excision of the latter. The implication of hypervariable rRNA d omains in apoptosis represents the first association of any functional process with these enigmatic parts of the ribosomes.