Messenger RNA in avian erythroblasts at the transcriptional and translational levels and the problem of regulation in animal cells

Abstract

The RNA metabolism in immature duck erythrocytes has been investigated in order to determine the characteristics of messenger RNA (mRNA) in a highly differentiated animal cell. mRNA-like fractions were obtained from polysomes, on the one hand, and from pulse-labeled total cells or isolated nuclei, on the other, and were characterized by sedimentation, labeling kinetics, base composition, and hybridization to homologous DNA. At the translational level, the pulse-labeled RNA from polysomes consists of a predominant species sedimenting with about 9S and of a class of polydisperse material sedimenting between 6 and 28S. Very little ribosomal RNA (rRNA) is synthesized. The 9S RNA has been purified. Its base composition is relatively high in G + C (but different from rRNA or transfer RNA) – as determined, after alkaline hydrolysis, by 32P distribution or spectrophotometric analysis. The polydisperse RNA has a base composition characterized by relatively high proportions of U and A and is similar in this respect to nuclear RNA. Total polysomal RNA hybridizes to homologous DNA. The biological activity tested in a cell-free protein-synthesizing system of Escherichia coli is highest in the 16 to 18S zone of polysomal RNA. The rapidly labeled RNA synthesized at the transcriptional level in the nuclei sediments predominantly in the 30 to 80S zone. Base-composition analysis of this RNA reveals the presence of a predominant fraction of high-U-type RNA and of a small amount of 45 and 32S rRNA precursors. The former fraction — tentatively termed nascent, messenger-like RNA (nascent mlRNA), with respect to its base composition and capacity of selective hybridization — is metabolically more unstable than the precursor rRNA. Hybridization experiments demonstrate that up to 7% of the DNA is homologous to the nascent RNA fractions. Polysomal RNA hybridizes to a much smaller extent and competes only slightly with the heavy nuclear fractions. The significance of this heavy, nascent mlRNA and its eventual role in the regulation of protein synthesis in animal cells is discussed. We conclude that in a highly differentiated cell many more mRNA species are produced than would be expressed phenotypically through protein synthesis in the polysome. A surprisingly large part of the genome is activated, but an important fraction of the transcription products never reaches the sites of protein synthesis. Thus, the spectrum of functional RNA is not defined through synthesis only, but is restricted during metabolism. Under these conditions, control of differentiation is probably not limited exclusively to the transcription of the genome, but is subject to regulation mechanisms operating at the intermediate or translational level.