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Fine mapping of 28S rRNA sites specifically cleaved in cells undergoing apoptosis
G Houge1, B Robaye, T S Eikhom
1Department of Anatomy and Cell Biology, University of Bergen, Norway.
Abstract:
Bona fide apoptosis in rat and human leukemia cells, rat thymocytes, and bovine endothelial cells was accompanied by limited and specific cleavage of polysome-associated and monosome-associated 28S rRNA, with 18S rRNA being spared. Specific 28S rRNA cleavage was observed in all instances of apoptotic death accompanied by internucleosomal DNA fragmentation, with cleavage of 28S rRNA and of DNA being linked temporally. This indicates that 28S rRNA fragmentation may be as general a feature of apoptosis as internucleosomal DNA fragmentation and that concerted specific cleavage of intra- and extranuclear polynucleotides occurs in apoptosis. Apoptosis-associated cleavage sites were mapped to the 28S rRNA divergent domains D2, D6 (endothelial cells), and D8. The D2 cuts 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 limited D2 cleavage but did produce a more generalized cleavage in the D8 region. Of potential functional interest was the finding that the primary cuts in D2 exactly flanked a 0.3-kb hypervariable subdomain (D2c), allowing excision of the latter. The implication of hypervariable rRNA domains in apoptosis represents the first association of any functional process with these enigmatic parts of the ribosomes.
Insights
Apoptosis involves specific cleavage of 28S ribosomal RNA (rRNA), similar to DNA fragmentation. This targeted rRNA breakdown, particularly in divergent domain 2 (D2), may facilitate the excision of hypervariable rRNA subdomains during programmed cell death.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is a fundamental biological process.
- Internucleosomal DNA fragmentation is a hallmark of apoptosis.
- The role of ribosomal RNA (rRNA) cleavage during apoptosis is not fully understood.
Purpose of the Study:
- To investigate the cleavage of 28S ribosomal RNA (rRNA) during apoptosis in various cell types.
- To determine if 28S rRNA cleavage is a general feature of apoptosis and temporally linked to DNA fragmentation.
- To map the specific cleavage sites within the 28S rRNA and explore their functional implications.
Main Methods:
- Analysis of rRNA cleavage in apoptotic rat and human leukemia cells, rat thymocytes, and bovine endothelial cells.
- Comparison of rRNA cleavage patterns in apoptosis versus necrotic cell death.
- Mapping of 28S rRNA cleavage sites using specific domains (D2, D6, D8).
Main Results:
- Specific and limited cleavage of 28S rRNA, but not 18S rRNA, was observed in all apoptotic cells studied.
- 28S rRNA cleavage was temporally linked to internucleosomal DNA fragmentation.
- Cleavage sites were mapped to divergent domains D2, D6, and D8, with D2 cuts flanking a hypervariable subdomain (D2c).
- Necrotic cell death did not induce specific D2 cleavage, suggesting apoptosis-specific mechanisms.
Conclusions:
- 28S rRNA fragmentation is a general feature of apoptosis, akin to DNA fragmentation.
- Apoptosis involves concerted cleavage of both intra- and extranuclear polynucleotides.
- The specific cleavage of 28S rRNA in apoptosis may allow for the excision of hypervariable rRNA domains, suggesting a functional role for these regions in programmed cell death.