Mammalian epidermal messenger RNA: identification and characterization of the keratin messengers

A messenger RNA fraction which directs the synthesis of epidermal keratins and other skin proteins has been isolated from adult guinea pigs and newborn rats, utilizing techniques designed to minimize degradation by endogenous nucleases. During the initial extraction procedures an inhibitor of translation was identified. This inhibitor could be removed by sedimentation of the RNA through cesium chloride. Electrophoresis of the resulting RNA on denaturing agarose/urea gels indicated that, in addition to 18S and 28S ribosomal RNAs, several minor species ranging in size from 10S to 28S were present. This heterogeneous RNA stimulated the incorporation of radioactive amino acids into protein in a reticulocyte lysate system which had an absolute dependence on added mRNA. A fraction of the RNA was retained on oligo(dT)-cellulose, indicating the presence of poly(adenylic acid) sequences. This poly(A)-rich material was considerably enriched for mRNA activity. Analysis of the products of synthesis indicated that proteins which migrated as keratins in 1- and 2-dimensional electrophoretic systems were major translation products of both the unfractionated material and poly(A)-containing fractions. The minimum sedimentation coefficient of keratin mRNAs was found to be 18S, a value consistent with the molecular weight of the keratin polypeptides.