Localization and structure of endonuclease cleavage sites involved in the processing of the rat 32S precursor to ribosomal RNA.
The initial endonuclease cleavage site in 32 S pre-rRNA (precursor to rRNA) is located within the rate rDNA sequence by S1-nuclease protection mapping of purified nucleolar 28 S rRNA and 12 S pre-rRNA. The heterogeneous 5'- and 3'-termini of these rRNA abut and map within two CTC motifs in tSi2 (internal transcribed spacer 2) located at 50-65 and 4-20 base-pairs upstream from the homogeneous 5'-end of the 28 S rRNA gene. These results show that multiple endonuclease cleavages occur at CUC sites in tSi2 to generate 28 S rRNA and 12 S pre-rRNA with heterogeneous 5'- and 3'-termini, respectively. These molecules have to be processed further to yield mature 28 S and 5.8 S rRNA. Thermal-denaturation studies revealed that the base-pairing association in the 12 S pre-rRNA:28 S rRNA complex is markedly stronger than that in the 5.8 S:28 S rRNA complex. The sequence of about one-quarter (1322 base-pairs) of the 5'-part of the rat 28 S rDNA was determined. A computer search reveals the possibility that the cleavage sites in the CUC motifs are single-stranded, flanked by strongly base-paired GC tracts, involving tSi2 and 28 S rRNA sequences. The subsequent nuclease cleavages, generating the termini of mature rRNA, seem to be directed by secondary-structure interactions between 5.8 S and 28 S rRNA segments in pre-rRNA. An analysis for base-pairing among evolutionarily conserved sequences in 32 S pre-rRNA suggests that the cleavages yielding mature 5.8 S and 28 S rRNA are directed by base-pairing between (i) the 3'-terminus of 5.8 S rRNA and the 5'-terminus of 28 S rRNA and (ii) the 5'-terminus of 5.8 S rRNA and internal sequences in domain I of 28 S rRNA. A general model for primary- and secondary-structure interactions in pre-rRNA processing is proposed, and its implications for ribosome biogenesis in eukaryotes are briefly discussed.
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D002466:Cell Nucleolus / Q000032:analysis; D003593:Cytoplasm / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases; D008099:Liver / Q000032:analysis; D008956:Models, Chemical; D009691:Nucleic Acid Denaturation; D009694:Nucleic Acid Precursors / Q000302:isolation & purification; D012322:RNA Precursors; D012335:RNA, Ribosomal / Q000302:isolation & purification; D051381:Rats; D015719:Single-Strand Specific DNA and RNA Endonucleases
D009694:Nucleic Acid Precursors; D012322:RNA Precursors; D012335:RNA, Ribosomal; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases