Staphylococcal nuclease digestion of relatively stable RNA in the chromatin and nuclear ribonucleoprotein fractions of human carcinoma cells

The organization of relatively stable nuclear RNA in the chromatin and nuclear ribonucleoprotein (RNP) fractions isolated from a human carcinoma cell line differed from that of the rapidly turning over RNA. In both fractions, the RNA labeled over 24 h was of a similar size as 1-h pulse-labeled RNA, but was digested more slowly and less extensively, although in all cases, the oligonucleotides protected from digestion were approximately 26 nucleotides in length. In addition, in the RNP fraction, RNA labeled over 24 h was in structures having a slightly higher bouyant density in CsCl than the structures containing the 1-h pulse-labeled RNA. A more stable class of discrete low molecular weight (lmw. 22,000 to 76,000) RNA species was also identified in the chromatin and RNP fractions. These species incorporated very little radioactivity over 1 or 24 h (or even 4 days). They cannot, therefore, account for the differences in digestion between 1-h and 24-h labeled RNA and represent a separate class. They (as well as the heterogenous nuclear RNA (hnRNA)) could be labeled by incorporation of [³H]methyl from methyl methionine into the base and 2'O-ribose positions and into the 'cap' structure. In the chromatin fraction, 90 to 100% of the methyl incorporated into RNA was protected from staphylococcal nuclease digestion, although the Imw RNA species were extensively nicked by the enzyme and the higher molecular weight methylated hnRNA was digested to smaller oligonucleotides. In the RNP fraction, only 40 to 60% of the methylated nucleotides were protected, and all of this was in small oligonucleotides, except for one Imw species at 37K. The protected RNA from both fractions contained all three methyl-labeled structures indentified in the intact RNA.