Transcriptional and post-transcriptional regulation in duck erythroblasts

Abstract

In order to study the relative importance of transcriptional and post-transcriptional regulation in duck erythroblasts, unique sequence pre-mRNA and mRNA complexity was assayed at four levels. Hybridization and Cr 0t analysis of total nuclear RNA with nick-translated unique sequence genomic DNA showed that of the order of 10% of the duck genome is transcribed; about one-half of the spectrum of RNA sequence formed decays within 40 min in the nucleus. The full-sequence complexity of cytoplasmic mRNA amounts to less than 0.15% of that of the genomic DNA. To assay for cytoplasmic controls, messenger RNA was isolated from polyribosomes and from repressed mRNP free of ribosomes; these two compartments contain 88 and 12%, respectively, of the polyadenylated mRNA. Both mRNA populations were either transcribed into cDNA by RNA-dependent DNA-polymerase, or translated in the nuclease-treated reticulocyte lysate. The kinetic complexity of the polyribosomal cDNA hybridizing back to its template shows the presence of about 200 mRNA species in three abundancy classes. The α- and β-globin mRNAs (70% in mass of polyribosomal mRNA) are represented by about 3800 copies/cell, 7 other mRNA types by about 90 copies/cell, and 202 mRNA by 2 copies/cell. The repressed free cytoplasmic mRNP contains 1400 different mRNAs, also in three abundancy classes. The α- and β-globin mRNAs (44% in mass) are represented by about 285 copies, there are 19 mRNA types with 7 copies, and 1380 mRNAs represented by less than 1 copy per cell. These data indicate that about 1200 mRNA species are not represented at all in polyribosomal mRNA. Heterologous cross-hybridization of cDNA transcribed from free mRNP message with polyribosomal mRNA suggests that the intermediate frequency mRNA classes are very differently represented in the two compartments. The qualitative difference of these mRNA populations is also demonstrated by the analysis of the products of in vitro translation by two-dimensional electrophoresis. Among the almost fully repressed mRNA is found, e.g., the mRNA for the polyribosomal poly(A)-binding protein. In conclusion the transcribed unique sequence DNA is of a complexity more than 100 times larger than that of total cytoplasmic mRNA; at an estimated mRNA/IVS ratio in pre-mRNA of 1:5 this amounts to a selection for transport to the cytoplasm of at best 1 mRNA molecule in 20. The repressed mRNA population in the cytoplasm is at least sevenfold more complex than the translated one. Individual equilibria of activity exist for each mRNA which allow almost full translation, and thus expression of some genes, but condition also full repression at post-transcriptional and cytoplasmic levels of a majority of the activated genes. These data show the higher importance of post-transcriptional controls compared to transcriptional regulation (selection of only 1 in 10) in a highly differentiated cell and are discussed in relation to the Cascade Regulation Scheme.