Pre-mRNA from erythroid enriched bone marrow cells of the rabbit. II. Characterization of pre-mRNA isolated by phenol extraction and poly(U)-Sepharose chromatography.

Precursor mRNA (pre-mRNA) was extracted from erythroid enriched bone marrow cells of the rabbit by the methods of Georgiev and Mantieva modified by Markov and Arion and of Holmes and Bonner, respectively. Density gradient centrifugation, base analysis and the effects of alpha-amanitin and actinomycin D on the synthesis of the cellular RNA showed signs of degradation in the rRNA-free 85 degrees C-fraction of the preparation according to Georgiev and Mantieva and a substantial rRNA contamination of the 65 degrees C-fraction. This RNA-fraction as well as the total RNA-preparation extracted according to Holmes and Bonner was purified from rRNA by affinity chromatography on poly(U)-Sepharose. Poly(A)+-RNA of all size-classes, among it a substantial amount of high molecular weight RNA (greater than 45 S), was isolated by this purification procedure. Especially the extraction according to Holmes and Bonner yields high molecular weight material but the critical step of this procedure often resulting in degradation of the RNA is the DNase treatment of the heavily DNA-contaminated total RNA-preparation either due to RNase contamination of the DNase or to the existence of RNase in the less intensive deproteinized RNA. The investigated cellular system is characterized by a very intensive rRNA synthesis which is typical for cells in the early stages of hematopoiesis. In contrast to investigations with purified RNA-polymerases and subcellular systems, but in accordance with data of in vivo experiments, alpha-amanitin inhibits both the pre-mRNA and the pre-rRNA synthesis.