Epidermal keratin messenger RNAs: A heterogeneous family

Abstract

A messenger RNA fraction from guinea-pig skin sediments on sucrose gradients at approx. 19 S and codes for keratin polypeptides in a messenger-dependent reticulocyte lysate system. Partial purification of this fraction was achieved by two cycles of chromatography on oligo(dT)-cellulose, followed by two cycles of sucrose gradient centrifugation. The identities of the protein products as keratins were established by co-electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, by peptide mapping, and by co-electrophoresis on two-dimensional polyacrylamide gels. All of the epidermal keratin polypeptides which are present in vivo are synthesized in vitro under the direction of this messenger. Fractionation of the messenger indicates that each different polypeptide is the product of a single mRNA species, and that no keratin is formed by proteolytic processing of higher molecular weight species or by polymerization of smaller precursors. Post-translational changes such as phosphorylation, which are known to occur in vivo, cannot be identified in the reticulocyte lysate system. Translation of these keratin messenger species is strongly inhibited by 7-methylguanosine 5′-triphosphate, indicating that the molecules have a ‘capped’ 5′-terminus.