Isolation and characterization of hnRNA-snRNA-protein complexes from Morris hepatoma cells.
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 less than 30 S to greater than 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 54 000-59 000. Incubation of the nuclear extracts at 0 C with low doses of pancreatic R Nase (up to 1.5 micrograms/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 micrograms/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.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008297:Male; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D009698:Nucleoproteins / Q000302:isolation & purification*; D012313:RNA / Q000302:isolation & purification; D012332:RNA, Heterogeneous Nuclear / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000302:isolation & purification*; D012342:RNA, Small Nuclear; D051381:Rats; D011919:Rats, Inbred Strains; D012260:Ribonucleases / Q000494:pharmacology; D012261:Ribonucleoproteins / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000494:pharmacology
D009363:Neoplasm Proteins; D009698:Nucleoproteins; D012332:RNA, Heterogeneous Nuclear; D012334:RNA, Neoplasm; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D012965:Sodium Chloride; D012313:RNA; D012260:Ribonucleases