Isolation and Characterization of hnRNA-snRNA-Protein Complexes from Morris Hepatoma Cells

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Abstract
Of the RNA labelled after incubation of hepatoma cells with radioactive precursors for 20 and 150 min, 35%, and 70%, respectively, can be isolated from nuclei by two consecutive extractions with 0.14 M NaCl at pH 8. The isolated RNA is complexed with nuclear proteins forming structures with sedimentation coefficients of < 30 S to > 100 S. Similar complexes from rat liver isolated under the same experimental conditions show coefficients of 30–40 S. The RNA‐associated proteins are similar, on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis, to the respective proteins of other cell types. The presence on these RNP complexes of six discrete small nuclear RNAs (snRNA) has been established. Experiments with a reversible inhibitor of RNA synthesis, d‐galactosamine, demonstrated differences in the turnover of hnRNA and snRNA. The half‐lives of the six snRNA species has been determined, varying from 32 h for snRNA species a, b and d, to 22 h for snRNA species e and f and to 13 h for snRNA species c. Treatment of the nuclear extracts with 0.7 M and 1 M NaCl results in dissociation of hnRNA from the ‘core’ and other polypeptides, whereas snRNA remains complexed with polypeptides of Mr 54000–59000. Incubation of the nuclear extracts at 0°C with low doses of pancreatic RNase (up to 1.5 μg/ml), which renders approximately 80% of the hnRNA acid‐soluble and cleaves most of the snRNA, results in conversion of the high‐molecular‐weight hnRNPs to 30‐S structures, without disrupting the 30‐S RNP. Treatment of the nuclear extracts with higher doses of RNase (3 μg/ml) leads to disruption of the 30‐S RNP and release of the hnRNA‐associated proteins, underlining the importance of hnRNA‐protein interaction for the retainment of the hnRNP structures.