Appearance of rapidly labeled, high molecular weight RNA in nuclear ribonucleoprotein. Release from chromatin and association with protein

Chromatin and nuclear ribonucleoprotein (nRNP) have been prepared from a human carcinoma cell line. Following a 1 hour [³H]uridine pulse, 60 to 70% of the nuclear radioactivity, after removal of nucleoli, was found in the chromatin, the balance in nRNP. This was true whether the chromatin and nRNP were separated by velocity centrifugation or by isopycnic centrifugation on Metrizamide gradients. Radioactivity in chromatin and nRNP was found in high molecular weight RNA, with mean sedimentation coefficients of 20 S and 15 S, respectively, as determined on sodium dodecyl sulfate sucrose gradients. Experiments on the kinetics of appearance of radioactivity in the RNA of the two fractions suggest that some of the chromatin associated RNA is precursor to nRNP RNA. The proteins of nRNP are complex as revealed by sodium dodecyl sulfate gel electrophoresis. The contamination by chromatin protein was estimated to be 5%. Experiments involving short pulses of [³H]tryptophan, and pulse chase, suggested that the rapidly turning over proteins of nRNP were not complexed with RNA while still associated with chromatin. However, it was also shown that the radioactivity in nRNP following short pulses of [³H]tryptophan did not correspond to the major bands seen on stained sodium dodecyl sulfate gels. It is therefore concluded that the protein of nRNP consists of two classes: species present in large amounts, possibly common to all RNA in nRNP, which are relatively stable and may be complexed to RNA still associated with chromatin; and a large number of rapidly turning over species, each present in small amounts and associated with nRNP only after its release from chromatin.