Comparisons of proteins associated with duck-globin mRNA and its polyadenylated segment in polyribosomal and repressed free messenger ribonucleoprotein complexes

Abstract

EDTA dissociation of polyribosomes from duck erythroblasts allowed us to isolate the 15-S globin messenger ribonucleoproteins (mRNP) by sucrose gradient centrifugation or affinity chromatography on poly(U)-Sepharose or oligo(dT)-cellulose columns. Their protein composition was compared by one and two-dimensional electrophoresis in sodium dodecyl sulfate to the free 20-S mRNP containing the repressed fraction of globin mRNA [Vincent, A., Civelli, O., Maundrell, K., and Scherrer, K. (1980) Eur. J. Biochem. 112, 617--633]. The protein composition of the 15-S mRNP isolated by these methods in different ionic strength conditions, was characterized by a major 73 000-Mr polypeptide and seven minor polypeptides with Mr ranging from 45 000 to 68 000, all of which are slightly basic, and about five acidic ones in the 80 000--130 000-Mr range. All these are retained in the 15-S mRNP core particle isolated at 0.5 M KCl. At low ionic strength, in addition, a specific group of acidic polypeptides in the Mr range 35 000--105 000 was also found associated with globin mRNA. Oligo(dT)-cellulose chromatography of mRNP digested with ribonucleases A and T1 indicated that the 73 000-Mr major protein is bound to the poly(A) segment; some other proteins resolved as minor components interact with both the poly(A) and non-poly(A) regions of globin mRNA. Characterization of proteins interacting with the poly(A) segment of non-polyribosomal globin mRNA in 20-S free mRNP demonstrated the absence of the polyribosomal 73 000-Mr poly(A)-binding protein. Furthermore, it confirmed that the protein compositions of translatable polyribosomal and repressed free globin mRNP are very different. Indeed, the respective core (0.5 M KCl) particles contain only two possibly common polypeptides. The specificity of proteins associated with globin mRNA in two different functional states shown here supports the hypothesis of a role of mRNP proteins in translational control of mRNA.