Synthesis of messenger-like RNA in avian erythrocyte nuclei

Abstract

Cell ghosts have been prepared from mature chicken erythrocytes using 0.05% saponin. Such preparations are capable of incorporating label from [3H]UTP and provide a system, where the nucleus is permeable to nucleotides and macromolecules, for studying the low-level RNA synthesis characteristic of these cells. RNase A (50 micrograms/ml) eliminated all radioactivity binding to DE-81 filters, indicating that the product was RNA; and DNase (10 micrograms/ml) and actinomycin D (10 micrograms/ml) each inhibited UMP incorporation by 70%, suggesting that the synthesis was DNA-dependent. Polymerization was inhibited 90% by 0.1 microgram/ml alpha-amanitin, and maximum synthesis occurred in the presence of high salt (0.175 M KCl) and Mn2+ (0.5 mM). Polyacrylamide gel electrophoresis indicated that the newly synthesized RNA was heterogeneous in size, having a distribution from 5 to 60 S with a significant fraction migrating as 8-12 S. Approximately 15% of the total RNA was bound by an oligo(dT)-cellulose column, suggesting that some RNA processing was occurring, although attempts to detect the incorporation of label from [alpha-32P]GTP into a 5'-cap structure were unsuccessful. In comparison to RNA synthesis in reticulocyte nuclei, both the rate and extent of transcription in erythrocyte nuclei were much reduced. Moreover, about 25-30% of the reticulocyte nascent RNA was released from the nuclei during a 60-min incubation, while no release was observed for the erythrocyte nuclei. Hybridization of radiolabeled RNA to excess chicken DNA indicated that the majority (80%) of the in vitro transcripts were complementary to unique sequence DNA (C0t1/2 = 4.5 X 10(3)). When RNA synthesized by either erythrocyte or reticulocyte nuclei was hybridized to cDNA complementary to reticulocyte polysomal mRNA, about 8% of the reticulocyte nuclear RNA but less than 1% of the erythrocyte nuclear RNA were resistant to RNase A digestion. Taken together, these data suggest that nuclei prepared by saponin lysis of chicken erythrocytes synthesize messenger-like RNA via endogenous polymerase II activity. A fraction of this RNA is polyadenylated but contains few, if any, globin sequences or other transcripts found on reticulocyte polysomes.