Release of ribonucleoprotein particles containing rapidly labeled ribonucleic acid from rat liver nuclei: Effect of adenosine 5′-triphosphate and some properties of the particles

Abstract

Nuclei labeled with [6- 14C]orotic acid for 40 min in vivo were isolated from rat liver by the modified method of Chauveau et al. 13. Release of rapidly labeled RNA from nuclei was studied by incubation with cytoplasmic ribonuclease inhibitor in vitro. 1. 1. Release of rapidly labeled RNA from isolated nuclei is dependent upon the following factors. (a) Temperature and duration of the incubation: The release is directly proportional to temperature in the range between 10 and 37° in the presence of 5 mM Mg 2+ and 1 mM ATP. (b) The concentration of ATP in the reaction mixture: ATP specifically stimulates the release. GTP, CTP and UTP are less stimulative, and ADP and AMP have almost no effect on the release. (c) The concentrations of bivalent cations: All of the bivalent cations tested prevent the release. Alkaline earth metals, such as Mg 2+, Ca 2+ and Ba 2+, react antagonistically to ATP. EDTA inhibits the release somewhat. (d) The pH of the incubation: The release of rapidly labeled RNA is directly proportional to pH in the range between pH 6.0 and 8.0. 2. 2. On incubation with 5 mM Mg 2+ and 3 mM ATP at 20°, rapidly labeled RNA is released in the form of ribonucleoprotein particles having a predominant s value of 45 S. 3. 3. The particles have a low RNA/protein ratio (1:5.6) and a low buoyant density in CsCl (1.40). They are very sensitive to ribonuclease, but quite insensitive to deoxyribonuclease, deoxycholate treatment and EDTA treatment. 4. 4. The specific activity of the released RNA (incubation at 20° for 5 min) labeled with [ 14C]orotic acid for 40 min in vivo is 3.3 times higher than that of RNA from cytoplasmic 45-S particles. Total nuclear RNA has the highest specific activity, which is 5 times higher than that of released RNA. 5. 5. Analyses of base composition of RNA released from nuclei by ATP show a high A+U G+C ratio, both by the ultraviolet absorption method (1.05) and by the radioactivity of 32P-short labeling (1.17). Sedimentation profiles of rapidly labeled RNA in the released fraction show the main peak at 14 S with a wide distribution from 6 to 28 S. 6. 6. The mechanism of the release of rapidly labeled RNA from nuclei by ATP is discussed. On the basis of a comparison of the 45-S ribonucleoprotein particles released from nuclei by ATP with other nuclear ribonucleoprotein particles and cytoplasmic 45-S particles, it is concluded that 45-S ribonucleoprotein particles released by ATP constitute the main form in which messenger RNA appears during its transportation from the nucleus to the cytoplasm.