Complexity of the structure of particles containing heterogeneous nuclear RNA as demonstrated by ribonuclease treatment.
Brain ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNA) and a large number of polypeptides ranging from 23000 to 150000 molecular weight were treated with pancreatic and T1 ribonucleases at low (0.1 or 0.5 microng plus 5 or 25 units/ml), high (2 microng plus 100 units/ml) and very high (20 microng plus 1000 units/ml) concentrations. At low enzyme concentration, a large fraction of the particle material accumulated at 35-45 S. The accumulation was more marked for proteins in the 30000-38000 molecular weight range. Heterogeneous complexes containing a large spectrum of proteins with a characteristic distribution were disclosed between 60 and 200 S. At high ribonuclease concentration the total quantity of proteins at 35-45 S decreased, but the proteins of 30000-38000 molecular weight predominated. Heterogeneous complexes were also present. Finally at very high enzyme concentration, the particles were almost entirely hydrolyzed. Only a small amount of heterogeneous complexes subsisted at 30-190 S. The RNA content of the remaining ribonucleoprotein complexes as well as its size decreased upon ribonuclease treatment as shown by CsCl density determination and electrophoretic analysis. The results suggested that the stability of the complexes depended upon protein-protein as well as RNA-protein interactions. They also showed that sequences up to 200-300 nucleotides were protected by proteins against ribonuclease. The results were compatible with the existence of at least 3 constituents in the particles. The previously defined monoparticle population accumulating at 35-45 S was heterogeneous in respect to ribonuclease sensitivity and protein composition. The 30000-38000 molecular weight proteins accumulating at low ribonuclease and predominating afterwards were assumed to belong to monoparticles alpha. Monoparticles beta contained a larger range of proteins more easily released by the enzymes. The heterogeneous complexes were a third constituent whose relationship with the monoparticles war not established yet. A large fraction of the particle phosphoproteins was associated to these complexes. On the basis of our experiments in vitro it is assumed that monoparticle alpha can be preferentially isolated from the nuclei under conditions where endogeneous ribonuclease is high and (or) not inhibited. This might explain why, in certain cases, only one or a few proteins were described in nuclear particles instead of the complete set present in the native particles.
D000818:Animals; D001923:Brain Chemistry; D002467:Cell Nucleus / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D008970:Molecular Weight; D009698:Nucleoproteins; D009843:Oligoribonucleotides / Q000032:analysis; D012313:RNA / Q000302:isolation & purification; D051381:Rats; D012260:Ribonucleases; D012261:Ribonucleoproteins
D009698:Nucleoproteins; D009843:Oligoribonucleotides; D012261:Ribonucleoproteins; D012313:RNA; D012260:Ribonucleases