Complexity of the Structure of Particles Containing Heterogeneous Nuclear RNA as Demonstrated by Ribonuclease Treatment

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Abstract
Brain [rat] ribonucleoprotein particles containing heterogeneous nuclear RNA and a large number of polypeptides ranging from 23,000-150,000 MW were treated with pancreatic [EC 3.1.4.22] and T1 [EC 3.1.4.8] RNase at low (0.1 or 0.5 .mu.g plus 5 or 25 units/ml), high (2 .mu.g plus 100 units/ml) and very high (20 .mu.g 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 30,000-38,000 MW range. Heterogeneous complexes containing a large spectrum of proteins with a characteristic distribution were disclosed between 60 and 200 S. At high RNase concentration the total quantity of proteins at 35-45 S decreased, but the proteins of 30,000-38,000 MW 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 on RNase treatment as shown by CsCl density determination and electrophoretic analysis. The stability of the complexes probably depended on protein-protein as well as RNA-protein interactions. Sequences up to 200-300 nucleotides were protected by proteins against RNase. 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 RNase sensitivity and protein composition. The 30,000-38,000 MW proteins accumulating at low RNase 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 3rd constituent whose relationship with the monoparticles was not established yet. A large fraction of the particle phosphoproteins was associated with these complexes. On the basis of these experiments in vitro it is assumed that monoparticle .alpha. can be preferentially isolated from the nuclei under conditions where endogeneous RNase is high and (or) not inhibited. This might explain why, in certain cases, only 1 or a few proteins were described in nuclear particles instead of the complete set present in the native particles.