Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1980 Oct 1;87(1):65–71. doi: 10.1083/jcb.87.1.65

Uptake and utilization of mRNA by myogenic cells in culture

PMCID: PMC2110704  PMID: 7419601

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.

Full Text

The Full Text of this article is available as a PDF (672.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bester A. J., Kennedy D. S., Heywood S. M. Two classes of translational control RNA: their role in the regulation of protein synthesis. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1523–1527. doi: 10.1073/pnas.72.4.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhargava P. M., Shanmugam G. Uptake of nonviral nucleic acids by mammalian cells. Prog Nucleic Acid Res Mol Biol. 1971;11:103–192. doi: 10.1016/s0079-6603(08)60327-x. [DOI] [PubMed] [Google Scholar]
  4. Brinster R. L., Chen H. Y., Trumbauer M. E., Avarbock M. R. Translation of globin messenger RNA by the mouse ovum. Nature. 1980 Jan 31;283(5746):499–501. doi: 10.1038/283499a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Devlin R. B., Emerson C. P., Jr Coordinate regulation of contractile protein synthesis during myoblast differentiation. Cell. 1978 Apr;13(4):599–611. doi: 10.1016/0092-8674(78)90211-8. [DOI] [PubMed] [Google Scholar]
  6. Dimitriadis G. J. Cellular uptake of ribonucleic acids entrapped into liposomes. Cell Biol Int Rep. 1979 Sep;3(6):543–549. doi: 10.1016/0309-1651(79)90090-0. [DOI] [PubMed] [Google Scholar]
  7. Doetschman T. C., Dym H. P., Siegel E. J., Heywood S. M. Myoblast stored myosin heavy chain transcripts are precursors to the myotube polysomal myosin heavy chain mRNAs. Differentiation. 1980 Jun;16(3):149–162. doi: 10.1111/j.1432-0436.1980.tb01071.x. [DOI] [PubMed] [Google Scholar]
  8. Dym H. P., Kennedy D. S., Heywood S. M. Sub-cellular distribution of the cytoplasmic myosin heavy chain mRNA during myogenesis. Differentiation. 1979;12(3):145–155. doi: 10.1111/j.1432-0436.1979.tb01000.x. [DOI] [PubMed] [Google Scholar]
  9. Dym H., Turner D. C., Eppenberger H. M., Yaffe D. Creatine kinase isoenzyme transition in actinomycin D-treated differentiating muscle cultures. Exp Cell Res. 1978 Apr;113(1):15–21. doi: 10.1016/0014-4827(78)90082-4. [DOI] [PubMed] [Google Scholar]
  10. Enger M. D., Hanners J. L. Informosomal and polysomal messenger RNA. Differential kinetics of polyadenylation and nucleocytoplasmic transport in Chinese hamster ovary cells. Biochim Biophys Acta. 1978 Dec 21;521(2):606–618. doi: 10.1016/0005-2787(78)90302-7. [DOI] [PubMed] [Google Scholar]
  11. Gallagher R. E., Walter C. A., Gallo R. C. Uptake and aminoacylation of exogenous transfer RNA by mouse leukemia cells. Biochem Biophys Res Commun. 1972 Nov 1;49(3):782–792. doi: 10.1016/0006-291x(72)90479-2. [DOI] [PubMed] [Google Scholar]
  12. Geetha V., Gnanam A. An in vitro protein-synthesizing system with isolated chloroplasts of Sorghum vulgare. An alternate assay system for exogenous template RNA. J Biol Chem. 1980 Jan 25;255(2):492–497. [PubMed] [Google Scholar]
  13. Gette W. R., Heywood S. M. Translation of myosin heavy chain messenger ribonucleic acid in an eukaryotic initiation factor 3- and messenger-dependent muscle cell-free system. J Biol Chem. 1979 Oct 10;254(19):9879–9885. [PubMed] [Google Scholar]
  14. Gottlieb A. A. Lymphoid cell RNA's and immunity. Prog Nucleic Acid Res Mol Biol. 1973;13:409–465. doi: 10.1016/s0079-6603(08)60108-7. [DOI] [PubMed] [Google Scholar]
  15. Gurdon J. B., Lane C. D., Woodland H. R., Marbaix G. Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature. 1971 Sep 17;233(5316):177–182. doi: 10.1038/233177a0. [DOI] [PubMed] [Google Scholar]
  16. Gurdon J. B., Lingrel J. B., Marbaix G. Message stability in injected frog oocytes: long life of mammalian alpha and beta globin messages. J Mol Biol. 1973 Nov 5;80(3):539–551. doi: 10.1016/0022-2836(73)90421-x. [DOI] [PubMed] [Google Scholar]
  17. Heysood S. M., Rich A. In vitro synthesis of native myosin, actin, and tropomyosin from embryonic chick polyribosomes. Proc Natl Acad Sci U S A. 1968 Feb;59(2):590–597. doi: 10.1073/pnas.59.2.590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heywood S. M., Kennedy D. S., Bester A. J. Separation of specific initiation factors involved in the translation of myosin and myoglobin messenger RNAs and the isolation of a new RNA involved in translation. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2428–2431. doi: 10.1073/pnas.71.6.2428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jacherts D., Drescher J. Antibody response in rhesus monkeys and guinea pigs to inoculation with RNA derived from antigenically stimulated cell-free systems. J Immunol. 1970 Mar;104(3):746–752. [PubMed] [Google Scholar]
  20. Kennedy D. S., Heywood S. M. The role of muscle and reticulocyte initiation factor 3 on the translation of myosin and globin messenger RNA in a wheat germ cell-free system. FEBS Lett. 1976 Dec 31;72(2):314–318. doi: 10.1016/0014-5793(76)80994-5. [DOI] [PubMed] [Google Scholar]
  21. Koch G., Bishop J. M. The effect of polycations on the interaction of viral RNA with mammalian cells: studies on the infectivity of single- and double-stranded poliovirus RNA. Virology. 1968 May;35(1):9–17. doi: 10.1016/0042-6822(68)90300-0. [DOI] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Levine A. J., Torosian M., Sarokhan A. J., Teresky A. K. Biochemical criteria for the in vitro differentiation of embryoid bodies produced by a transplantable teratoma of mice. The production of acetylcholine esterase and creatine phosphokinase by teratoma cells. J Cell Physiol. 1974 Oct;84(2):311–317. doi: 10.1002/jcp.1040840217. [DOI] [PubMed] [Google Scholar]
  24. Lodish H. F., Jacobsen M. Regulation of hemoglobin synthesis. Equal rates of translation and termination of - and -globin chains. J Biol Chem. 1972 Jun 10;247(11):3622–3629. [PubMed] [Google Scholar]
  25. Lough J., Bischoff R. Differentiation of creatine phosphokinase during myogenesis: quantitative fractionation of isozymes. Dev Biol. 1977 Jun;57(2):330–344. doi: 10.1016/0012-1606(77)90219-6. [DOI] [PubMed] [Google Scholar]
  26. McLean M. J., Renaud J. F., Niu M. C., Sperelakis N. Membrane differentiation of cardiac myoblasts induced in vitro by an RNA-enriched fraction from adult heart. Exp Cell Res. 1977 Nov;110(1):1–14. doi: 10.1016/0014-4827(77)90263-4. [DOI] [PubMed] [Google Scholar]
  27. Morse R. K., Herrmann H., Heywood S. M. Extraction with Triton X-100 of active polysomes from monolayer cultures of embryonic muscle cells. Biochim Biophys Acta. 1971 Mar 11;232(2):403–409. doi: 10.1016/0005-2787(71)90593-4. [DOI] [PubMed] [Google Scholar]
  28. NIELSEN L., LUDVIGSEN B. Improved method for determination of creatine kinase. J Lab Clin Med. 1963 Jul;62:159–168. [PubMed] [Google Scholar]
  29. NIU M. C., CORDOVA C. C., NIU L. C. Ribonucleic acid-induced changes in mammalian cells. Proc Natl Acad Sci U S A. 1961 Oct 15;47:1689–1700. doi: 10.1073/pnas.47.10.1689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Niu M. C., Deshpande A. K. The development of tubular heart in RNA-treated post-nodal pieces of chick blastoderm. J Embryol Exp Morphol. 1973 Apr;29(2):485–501. [PubMed] [Google Scholar]
  31. Nudel U., Soreq H., Littauer U. Z. Globin mRNA species containing poly(A) segments of different lengths. Their functional stability in Xenopus oocytes. Eur J Biochem. 1976 Apr 15;64(1):115–121. doi: 10.1111/j.1432-1033.1976.tb10279.x. [DOI] [PubMed] [Google Scholar]
  32. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  33. OLIVER I. T. A spectrophotometric method for the determination of creatine phosphokinase and myokinase. Biochem J. 1955 Sep;61(1):116–122. doi: 10.1042/bj0610116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Poste G., Papahadjopoulos D., Vail W. J. Lipid vesicles as carriers for introducing biologically active materials into cells. Methods Cell Biol. 1976;14:33–71. doi: 10.1016/s0091-679x(08)60468-9. [DOI] [PubMed] [Google Scholar]
  35. Sakai T. T., Cohen S. S. Uptake and aminoacylation of exogenous Escherichia coli tRNA by mouse fibroblasts. Biochem Biophys Res Commun. 1977 Sep 23;78(2):539–546. doi: 10.1016/0006-291x(77)90212-1. [DOI] [PubMed] [Google Scholar]
  36. Stebbing N. Cellular uptake and in vivo fate of polynucleotides. Cell Biol Int Rep. 1979 Sep;3(6):485–502. doi: 10.1016/0309-1651(79)90085-7. [DOI] [PubMed] [Google Scholar]
  37. Swanson R. F. Incorporation of high molecular weight polynucleotides by isolated mitochondria. Nature. 1971 May 7;231(5297):31–34. doi: 10.1038/231031a0. [DOI] [PubMed] [Google Scholar]
  38. Tepperman K., Morris G., Essien F., Heywood S. M. A mechanical dissociation method for preparation of muscle cell cultures. J Cell Physiol. 1975 Dec;86(3 Pt 1):561–565. doi: 10.1002/jcp.1040860313. [DOI] [PubMed] [Google Scholar]
  39. Young R. B., Goll D. E., Stromer M. H. Isolation of myosin-synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion. Dev Biol. 1975 Nov;47(1):123–135. doi: 10.1016/0012-1606(75)90268-7. [DOI] [PubMed] [Google Scholar]
  40. de Maeyer-Guignard J., de Maeyer E., Montagnier L. Interferon messenger RNA: translation in heterologous cells. Proc Natl Acad Sci U S A. 1972 May;69(5):1203–1207. doi: 10.1073/pnas.69.5.1203. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES