Hybrid selection of messenger ribonucleoprotein for serum albumin: analysis of specific message-bound proteins.

Abstract

The messenger RNA for chicken serum albumin together with its specific binding proteins was purified by hybrid selection using filter-bound cloned albumin cDNA. Under conditions where hybridization of the protein-mRNA complex was specific for the immobilized cDNA sequence, no dissociation of the complex into its protein and RNA components was apparent. Electrophoretic analysis of albumin mRNA-specific binding proteins showed three major bands.Two of these were prominent in total poly(A) messenger ribonucleoprotein. The third band was of much greater relative intensity in the albumin mRNA-specific proteins than in total poly(A) messenger ribonudeoprotein. The results suggest that the proteins bound to albumin mRNA represent only a subset of the total population of poly(A) mRNA-associated protein.In eukaryotic cells, mRNA is found complexed with a variety of proteins and is more appropriately termed messenger ribonucleoprotein (mRNP) (1). Many investigators have shown that mRNPs are found both associated with polysomes (i.e., are being actively translated) and in the postpolysomal ("free") fraction (2). The relationship between free and polysome-associated mRNPs is not well understood and may differ from system to system. The protein components of mRNPs from many cell types have been analyzed, and in most cases differences are found between the free and polysome-associated fractions (3). Although no functions for proteins from mRNPs are known with certainty, with the exception of the poly(A) organizing protein (4), strong circumstantial evidence suggests that at least some of these proteins are accessory factors for protein synthesis or are involved in other aspects of translational regulation (5, 6).One outstanding difficulty in the study of mRNPs has been the inability, except in highly specialized systems, to analyze the proteins bound to a particular mRNA sequence. Thus, it is not known if the same mRNA sequence binds the same set of proteins in both the free and the polysome-associated fractions or, conversely, if different mRNA sequences in the same fraction bind the same set of proteins. This problem became important in our laboratory since we observed that the protein components of mRNPs ofimmature cockerel liver changed markedly in response to estradiol stimulation (7). Estradiol stimulation also profoundly alters the mRNA sequence population (8), rates of protein synthesis for specific proteins (9), and other aspects of hepatic metabolism. In this report, we show that one abundant chicken liver mRNP, that for serum albumin, may be purified by hybridization to cloned albumin cDNA sequences and that the proteins bound to it appear to be a limited subset ofthe population ofproteins associated with total mRNPs. MATERIALS AND METHODSLiver poly(A) mRNA was isolated from estradiol-injected cockerels according to Chirgwin et al. (10). Poly(A) mRNPs were isolated as described (7). cDNAs for chicken serum albumin and very low density lipoprotein II apoprotein (apoVLDL II) (a low molecular weight estradiol-...