Definition of subclasses of nucleoplasmic RNA

Abstract

RNA synthesis in isolated HeLa cell nuclei prepared from cells pretreated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is inhibited in a time-dependent manner. After 40-min pretreatment of cells with 60 muM DRB in the presence of actinomycin D (0.04 mug/ml), the rate of RNA synthesis in isolated nuclei, measured by [(3)H]UTP incorporation, is decreased by 63%. The DRB-resistant one-third of heterogeneous nuclear is distributed over the entire size range of heterogeneous nuclear RNA with some enrichment in the 18S range, as was observed earlier by pulse-labeling whole cells. A subclass of nucleoplasmic RNA molecules is defined in the approximate size range 110 to 250 x 10(3) daltons (330-740 nucleotides). By using heparin (2 mg/ml) to block the synthesis of smaller RNA, a peak in the chain-length range 330-740 nucleotides can be clearly resolved on 2.2% polyacrylamide/1% agarose gels in nuclei from control and DRB-treated cells. The synthesis of these molecules is largely ( approximately 90%) resistant to DRB but sensitive to alpha-amanitin at 1 mug/ml. The in vitro synthesis of molecules in the 140-330 residue range is also sensitive to alpha-amanitin at 1 mug/ml, and it is not at all affected by pretreatment of cells with DRB. Although the synthesis of the RNA in both the 330-740 and the 140-330 residue size ranges appears to be catalyzed by RNA polymerase II, the results with heparin suggest that there may be reinitiation of molecules in the 140-330 size range but not in the 330-740 range in vitro. The synthesis of 4.5S RNA ( approximately 100 nucleotides) and 5S RNA (120 nucleotides) is unaffected by pretreatment of cells with DRB and, as previously reported, is catalyzed by RNA polymerase III, with reinitiation occurring in vitro. Addition of DRB directly to isolated HeLa cell nuclei in vitro has no detectable effect on the overall rate of RNA synthesis.