Synthesis of Myosin Light Chains and Accumulation of Translatable mRNA Coding for Light Chain-like Polypeptides in Differentiating Muscle Cultures

Abstract

Rat skeletal muscle myosin contains small subunits (light chains, here designated LC1, LC2, LC3) of molecular weight 23,000, 17,000 and 15,000, respectively. The synthesis of myosin light chains during differentiation and the accumulation of mRNA which codes for these proteins were investigated in differentiating rat skeletal muscle cultures. When cultures were labeled prior to cell fusion, radioactive light chains which co-migrated with skeletal muscle myosin light chains on gel electrophoresis were absent or only barely detectable. The low molecular weight peptides which were associated with the heavy chain of myosin extracted from pre-fusion cultures differed in their electrophoretic mobility from light chains of skeletal muscle myosin. Following cell fusion, the amount of labeled LC1 and LC2 increased rapidly. The synthesis of LC3 was barely detectable during cell fusion and never exceeded one-fifth of the amounts of LC1 and LC2 synthesized. Polyadenylated RNA extracted at different times during differentiation was translated in the wheat germ cell-free system. The products were analyzed on isoelectnc focusing-SDS two-dimensional gel electrophoresis, and the radioactivity of the polypeptides co-migrating with myosin light chains was measured. Small amounts of radioactive products co-migrating with LC1 and LC2 became detectable among products of RNA preparations extracted several hours prior to cell fusion. However, the cell-free system directed by post-fusion RNA synthesized much larger amounts of LC1- and LC2- like polypeptides. Rapid accumulation of translatable mRNA for LC1 and LC2 was closely correlated with cell fusion. Radioactive polypeptides co-migrating with LC3 were synthesized in significant amounts in a cell-free system directed by pre-fusion RNA and increased only moderately when RNA extracted after fusion was used.