Isolation of a nuclear ribonucleoprotein network that contains heterogeneous RNA and is bound to the nuclear envelope.
Rapidly labeled polydispersed nuclear RNA is part of a ribonucleoprotein (RNP) network which in turn is tightly bound to the nuclear membrane. The membranous attachment, therefore, established a connection between chromatin and cytoplasm. The ultrastructure of the RNP network comprises fibrils and granules similar to those observed in intact nuclei. When bound to the nuclear membrane it has the composition of 63% protein, 14% RNA, 0.4% DNA, and 22.6% lipids. The proportion of lipids diminishes to 2.2% when nuclear membrane is not present. Chromatin, nucleoli, and ribosomes are minor contaminants since histones and ribosomal proteins are not detectable in polyacrylamide gel electrophoresis. Nuclear disruption at high pressure in a French pressure cell causes fragmentation of the RNP network into a series of polydispersed RNP particles. Fragmentation can be prevented by using mild pressure, or by disrupting nuclei with high salt buffer and digesting the dispersed chromatin with deoxyribonuclease. A RNP network, almost free of membrane, is also obtained if the nucleus is deprived of its envelope by treatment with Triton X-100. Since no polydispersed RNP particles are found following dissolution of the nuclear membrane, it is assumed that the particles are components of the RNP network whose fragmentation occurs as a consequence of two processes: (a) activation of nuclear nucleases and (b) shearing forces.
D000818:Animals; D002287:Carcinoma, Krebs 2 / Q000032:analysis; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000032:analysis* / Q000648:ultrastructure; D003851:Deoxyribonucleases; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000032:analysis; D008566:Membranes / Q000032:analysis; D051379:Mice; D008854:Microscopy, Electron; D009698:Nucleoproteins / Q000302:isolation & purification*; D012313:RNA / Q000302:isolation & purification*; D051381:Rats; D012261:Ribonucleoproteins / Q000302:isolation & purification*
D009698:Nucleoproteins; D012261:Ribonucleoproteins; D012313:RNA; D003851:Deoxyribonucleases