Identification and characterization of the translationally repressed cytoplasmic globin messenger-ribonucleoprotein particles from duck erythroblasts

Abstract

Globin messenger ribonucleoprotein (mRNP) particles which have been isolated from duck erythroblast post-polyribosomal supernatant are translationally inactive in vivo and in vitro but contain translatable mRNA active after deproteinisation. They were characterized following purification by successive sucrose gradient sedimentation in a buffer containing 0.05 M KCl. The complex, which sediments homogeneously at about 20 S, has a density of 1.39 g/cm3 and thus consists of four parts protein to one part RNA; 40% of this RNA is globin mRNA and no other mRNA could be detected. Sedimentation of the purified globin mRNP on sucrose gradients in 0.5 M KCl produced four components while polyacrylamide gel electrophoresis in non-denaturing conditions and in the presence of EDTA resulted in the separation of three components. Hybridization to globin cDNA and translation in vitro of the RNA extracted from these subparticles revealed the existence of two core particles containing globin mRNA with nominal sedimentation coefficients of 13 S and 16 S. Analysis of the protein components of the isolated sub-complexes by dodecyl sulfate and bidimensional gel electrophoresis indicated a very characteristic protein composition for each of these complexes. The 16-S and 13-S globin mRNPs differed essentially by the presence in the 13-S mRNP only of a group of major polypeptides. Of the other two sub-complexes, one consisted of 90% small RNA in the 4-S range; the second sedimented ahead of the globin mRNP core particles at about 19S and consisted of a very characteristic set of about 14 polypeptides. The polyribosomal 73000-Mr poly(A)-binding protein was not detected in the purified free globin mRNP although the mRNA in the untranslatable particle is polyadenylated. The presence in the cytoplasm of duck erythroblasts of two forms of untranslated globin messenger ribonucleoprotein particles, distinct in their protein composition from polyribosomal globin mRNP, suggests that they may have a specific role in the regulation of translation of globin mRNA.