Uptake and utilization of mRNA by myogenic cells in culture.

Abstract

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 .A number ofstudies has suggested that not only purified DNA but also purified RNA can be taken up by cells (reviewed in references 3, 14, and 38) . The first direct evidence arose from repeated observations of the infectivity of purified viral RNA and viroids (21,35). The aminoacylation ofbacterial tRNA by mouse fibroblasts in culture (37) and the ability of RNA extracted from human fibroblasts treated with poly(I) " poly(C) to elicit production of human interferon in chick fibroblasts as well as the production of mouse interferon by avian and simian cells incubated with mRNA extracted from mouse cells (26) serve to further demonstrate the uptake of functional RNA by cells.The cellular uptake of RNA has been reported to result in specific immune responses (19), the synthesis of specific proteins (30), as well as the induction ofmembrane differentiation (27,31). In most of the cases in which RNA has been demonstrated to enter cells, there is a lack of information concerning the manner by which it penetrates the cells and subsequently exerts its effects, and often the RNA itself has been poorly characterized. On the other hand, specific mRNA transcripts and their activities have been studied after entry into Xenopus

Encapsulation of "core" eIF3, regulatory components of eIF3 and mRNA into liposomes, and their subsequent uptake into myogenic cells in culture.

“…These experiments suggest that HAF has a discriminatory role in the intact cell . The fact that MHC-mRNA, inserted in myoblasts in the presence of HAF, was translated with a similar efficiency as previously observed in myotube cell cultures (17) suggests that HAF is a developmentally programmed regulatory protein appearing during myogenesis . These experiments lend strong support to our previous in vitro studies, suggesting that mRNA discriminatory elements play a significant role in the cytoplasmic utilization of mRNAs during muscle differentiation.…”

“…In this case, 24-h dividing myoblast cell cultures were used, as it is known that they contain little if any MHC-mRNA . In addition, we have demonstrated previously that MHC-mRNA can be translated in these cells, but much less efficiently than in differentiated myotubes (17). The position of [3H]MHC-mRNA when delivered alone and after preincubation with the HAF is displayed in Fig .…”

“…By inserting macromolecules into living cells and observing their effects and utilization a comparison of the results obtained from cell-free systems could be made. We have recently observed that deproteinized mRNA can be taken up by myogenic cells in culture and that MHC-mRNA is differentially translated in myoblasts as compared to myotubes (17). Recent advances in liposome technology, specifically with regard to the use of large unilamellar vesicles (LUV), allow the introduction of macromolecules into cells (22).…”

Induction of Somites by Myosin mRNA

“…The ability of the cells to take up and to correctly translate exogenously supplied mRNA had already been demonstrated in a critical review by Mroczkowski et al [28] of myoblast culture data. Somite induction in the presence of actinomycin D and its absence in the pres ence of puromycin in our experiments seems to indicate that translation of mRNA also occurs in our explants.…”

The virtosome—a novel cytosolic informative entity and intercellular messenger

“…RNA release from eukaryote cells has been demonstrated 48, 57–68. In initial studies, macromolecular RNA was shown to pass between 3T3 cells in vitro 57.…”