HETEROGENEITY OF THE NON-POLYSOMAL CYTOPLASMIC (FREE) MESSENGER-RNA . PROTEIN COMPLEXES OF EMBRYONIC CHICKEN MUSCLE

The distribution of pulse‐labeled poly(A)‐containing RNAs between the polysomal and nonpolysomal (free) cytoplasmic fractions of embryonic chicken muscle was examined by affinity chromatographys on oligo(dT)‐cellulose. Approximately 60% of the total cytoplasmic poly(A)‐containing RNAs were present in the polysome; the remainder were present outside the polysome, the 3H‐labeled ones being present in the form of ribonucleoprotein complexes. These free mRNA · protein complexes can be divided into two populations: one can bind to an oligo(dT)‐cellulose column as a ribonucleoprotein complex, suggesting that the 3′‐poly(A) region was available for interaction with the oligo(dT) residues; in the other, this region was presumably covered with proteins and can only bind to the oligo(dT)‐cellulose column following their removal. The mRNA · protein bound to oligo(dT)‐cellulose was subfractionated into three populations. The first fraction was eluted from the column by using low‐ionic‐strength buffer at 2°C, the major portion was eluted by a combination of low‐ionic‐strength buffer and a temperature of 37°C, while the remainder was eluted with 50% formamide. Each fraction displayed a distinctive buoyant density in a CsCl gradient, suggesting a difference in their protein content. The nature of polypeptides associated with these fractions was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Polypeptides of Mr 36500, 41000, 45000, 47000, 51000 and 75000 were common to all three mRNA protein fractions. The third population (eluted with 50% formamide) contains five additional polypeptides of Mr 54000, 66000, 68000, 73000 and 105000. The direct interaction of free proteins with oligo(dT)‐cellulose was studied by modification of the binding of mRNA · protein to oligo(dT) by saturation of oligo(dT) sequences with poly(A) or the poly(A) segment of mRNA. protein with poly(U). Two polypeptides of Mr 41 000 and 54000 showed independent binding to these columns; they were eluted with 50 formamide but not at 37°C in low‐salt buffer. Therefore, the source of the 41000‐Mr and 54000‐Mr polypeptides was considered as other than mRNA · protein. The translation of these mRNA · protein fractions showed that all three can be translated either as protein‐free RNA or complexed with proteins. Copyright © 1979, Wiley Blackwell. All rights reserved