Uptake and utilization of mRNA by myogenic cells in culture.

Primary chick myoblast cultures demonstrate the ability to take up exogenously supplied polyadenylated RNA and express the encoded information in a specific manner. This expression is shown to exhibit tissue specificity. Analysis of creatine kinase activity monitored at various times of incubation in the presence of either polyadenylated or nonpolyadenylated RNA indicates that only the poly(A)+ mRNA is capable of being actively translated. Radioactively labled poly(A)+ mRNA is taken up by the cell cultures in a time-dependent manner and subsequently shown to be associated with polysomes. This association with polysomes does not occur in the presence of puromycin and is unaffected by actinomycin D. Thus, nonspecific interaction with polysomes and induction of new RNA synthesis are ruled out and the association of the exogenously supplied poly(A)+ mRNA with polysomes is indicative of its translation in the recipient cells. When heterologous mRNA (globin) is supplied to the myoblasts, it is also taken up and properly translated. In addition, exogenously supplied myosin heavy chain mRNA is found associated with polysomes consisting of 4-10 ribosomes in myoblast cell cultures while in myotubes it is associated with very large polysomes, thus reflecting the different translational efficiencies that this message exhibits at two very different stages of myogenesis. The results indicate that muscle cell cultures can serve as an in vitro system to study translational controls and their roles in development.

Sub-cellular distribution of the cytoplasmic myosin heavy chain mRNA during myogenesis.

In the light of earlier work [1] which demonstrated the presence of a large number of myosin heavy chain (MHC) transcripts in chick myoblasts prior to cell fusion and the burst of MHC synthesis it was of great interest to determine the subcellular localization of the still inactive transcripts. It has been determined in differentiating muscle cells in culture. Two populations of cells were examined -- monucleated myoblasts just prior to cell fusion and myotubes where at least 80% of the cells were fused. Utilizing a myosin complementary DNA (cDNA) probe [2] it is observed that just prior to cell fusion, when the "burst" of myosin synthesis has not yet occurred, the vast majority of cytoplasmic myosin mRNA transcripts are found in a stored messenger RNA protein complex with a minimal amount found in the heavy polysome fraction. In differentiated myotube cultures, when myosin synthesis is progressing at a high rate, the reverse is found, i.e, the amount of stored myosin messenger RNA (mRNA) is minimal while the largest amount of myosin mRNA transcripts are localized in the polysome fraction. The number of total cytoplasmic myosin transcripts is found to decrease after cell fusion at a time when myosin synthesis is maximal suggesting that the efficiency of translation of myosin mRNA increases during terminal differentiation.

Myoblast stored myosin heavy chain transcripts are precursors to the myotube polysomal myosin heavy chain mRNAs.

Radioactively labeled myosin heavy chain messenger ribonucleic acid (MHC mRNA) synthesized during the pre-fusion stage of chick embryo breast muscle cell culture is transferred from messenger ribonucleic acid proteins (mRNPs) to the polysomal MHC mRNA during the period of rapid increase in the rate of MHC synthesis (mid-to late-fusion). This transfer constitutes a major contribution to the rate of incorporation of 3H-labeled transcripts into polysomal MHC mRNA at this time. As the increase in the rate of MHC synthesis levels off (late-to post-fusion) the contribution to the rate of incorporation of 3H-labeled transcripts into polysomal MHC mRNA from newly synthesized transcripts increases until it becomes predominant. In vivo, the level of MHC mRNP increases during early stages of embryonic development and then decreases when MHC synthesis and the level of polysomal MHC mRNA has been shown to increase.