Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNAin situ

Abstract

The secondary structure of mouse Ehrlich ascites 18S, 5.8S and 28S ribosomal RNA in situ was investigated by chemical modification using dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulphonate. These reagents specifically modify unpaired bases in the RNA. The reactive bases were localized by primer extension followed by gel electrophoresis. The three rRNA species were equally accessible for modification i.e. approximately 10% of the nucleotides were reactive. The experimental data support the theoretical secondary structure models proposed for 18S and 5.8/28S rRNA as almost all modified bases were located in putative single-strand regions of the rRNAs or in helical regions that could be expected to undergo dynamic breathing. However, deviations from the suggested models were found in both 18S and 28S rRNA. In 18S rRNA some putative helices in the 5'-domain were extensively modified by the single-strand specific reagents as was one of the suggested helices in domain III of 28S rRNA. Of the four eukaryote specific expansion segments present in mouse Ehrlich ascites cell 28S rRNA, segments I and III were only partly available for modification while segments II and IV showed average to high modification.

Fine Mapping of 28S rRNA Sites Specifically Cleaved in Cells Undergoing Apoptosis

“…Similar observations were made with mouse 28S rRNA, in which the U's corresponding to the U's involved in apoptotic D8 cleavage (Fig. 7C) were highly accessible to chemical modification (20). In addition, the 3Ј bulge loop that is cleaved in D8 has been found to be prone to RNase T 2 digestion in studies done with in vitro-transcribed human D8b regions (30).…”

“…Neither such extracts nor selected added RNases could reproduce the apoptosis-specific cuts in D2, both of which occur in regions recently shown to be inaccessible to chemical modification in the isolated ribosome (18,20). These findings indicate the existence of apoptosisassociated changes of ribosome conformation.…”

“…The discrete nature of the observed cleavages argues against generally increased RNase activity or random polynucleotide degradation as the explanation for apoptotic rRNA fragmentation. Another argument is that the regions cleaved in the D2 subdomain are known to be inaccessible to chemical modification in the intact ribosome (18,20), suggesting that the ribosome conformation is changed during apoptosis in such a way as to expose these D2c regions to RNase. We believe that 28S rRNA fragmentation is an example of the limited, selective, and controlled macromolecular degradation characteristic of apoptotic cells before secondary necrosis occurs (23,24,36).…”

“…In addition, the 3Ј bulge loop that is cleaved in D8 has been found to be prone to RNase T 2 digestion in studies done with in vitro-transcribed human D8b regions (30). Apparently, in the D8b tertiary structure this bulge loop is accessible to RNases (30), and the D8 region itself is exposed on the ribosomal surface (18,20). When the apoptotic D8 cleavage sites are compared, the consensus sequence is GCGGU ê U(U)CCC.…”

Diffusion-based Transport of Nascent Ribosomes in the Nucleus

“…Oligo 4 is targeted to a region of the large highly variable expansion sequence D2 (Gerbi, 1996), where insertion of a marker sequence in Tetrahymena is known to be viable (Sweeney et al, 1996). Oligo 5 is targeted to a region in the expansion sequence D12 that is known to be available for chemical modification in mouse ribosomes, indicating that this region may be uncovered near the surface of the ribosome (Holmberg et al, 1994a). Also, an insert at this site is viable in Tetrahymena (Sweeney et al, 1996).…”