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. 1993 Oct 2;123(2):431–441. doi: 10.1083/jcb.123.2.431

Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes

PMCID: PMC2119827  PMID: 7691830

Abstract

We have studied transport and localization of MBP mRNA in oligodendrocytes in culture by microinjecting labeled mRNA into living cells and analyzing the intracellular distribution of the injected RNA by confocal microscopy. Injected mRNA initially appears dispersed in the perikaryon. Within minutes, the RNA forms granules which, in the case of MBP mRNA, are transported down the processes to the periphery of the cell where the distribution again becomes dispersed. In situ hybridization shows that endogenous MBP mRNA in oligodendrocytes also appears as granules in the perikaryon and processes and dispersed in the peripheral membranes. The granules are not released by extraction with non-ionic detergent, indicating that they are associated with the cytoskeletal matrix. Three dimensional visualization indicates that MBP mRNA granules are often aligned in tracks along microtubules traversing the cytoplasm and processes. Several distinct patterns of granule movement are observed. Granules in the processes undergo sustained directional movement with a velocity of approximately 0.2 micron/s. Granules at branch points undergo oscillatory motion with a mean displacement of 0.1 micron/s. Granules in the periphery of the cell circulate randomly with a mean displacement of approximately 1 micron/s. The results are discussed in terms of a multi-step pathway for transport and localization of MBP mRNA in oligodendrocytes. This work represents the first characterization of intracellular movement of mRNA in living cells, and the first description of the role of RNA granules in transport and localization of mRNA in cells.

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Selected References

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  1. Barbarese E. Spatial distribution of myelin basic protein mRNA and polypeptide in quaking oligodendrocytes in culture. J Neurosci Res. 1991 Jul;29(3):271–281. doi: 10.1002/jnr.490290302. [DOI] [PubMed] [Google Scholar]
  2. Berleth T., Burri M., Thoma G., Bopp D., Richstein S., Frigerio G., Noll M., Nüsslein-Volhard C. The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo. EMBO J. 1988 Jun;7(6):1749–1756. doi: 10.1002/j.1460-2075.1988.tb03004.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Biegel D., Pachter J. S. "In situ" translation: use of the cytoskeletal framework to direct cell-free protein synthesis. In Vitro Cell Dev Biol. 1991 Jan;27(1):75–85. doi: 10.1007/BF02630897. [DOI] [PubMed] [Google Scholar]
  4. Bottenstein J. E. Growth requirements in vitro of oligodendrocyte cell lines and neonatal rat brain oligodendrocytes. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1955–1959. doi: 10.1073/pnas.83.6.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Braun R. E., Peschon J. J., Behringer R. R., Brinster R. L., Palmiter R. D. Protamine 3'-untranslated sequences regulate temporal translational control and subcellular localization of growth hormone in spermatids of transgenic mice. Genes Dev. 1989 Jun;3(6):793–802. doi: 10.1101/gad.3.6.793. [DOI] [PubMed] [Google Scholar]
  6. Bruckenstein D. A., Lein P. J., Higgins D., Fremeau R. T., Jr Distinct spatial localization of specific mRNAs in cultured sympathetic neurons. Neuron. 1990 Dec;5(6):809–819. doi: 10.1016/0896-6273(90)90340-l. [DOI] [PubMed] [Google Scholar]
  7. Burgin K. E., Waxham M. N., Rickling S., Westgate S. A., Mobley W. C., Kelly P. T. In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain. J Neurosci. 1990 Jun;10(6):1788–1798. doi: 10.1523/JNEUROSCI.10-06-01788.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Colman D. R., Kreibich G., Frey A. B., Sabatini D. D. Synthesis and incorporation of myelin polypeptides into CNS myelin. J Cell Biol. 1982 Nov;95(2 Pt 1):598–608. doi: 10.1083/jcb.95.2.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ephrussi A., Dickinson L. K., Lehmann R. Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell. 1991 Jul 12;66(1):37–50. doi: 10.1016/0092-8674(91)90137-n. [DOI] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  11. Fontaine B., Sassoon D., Buckingham M., Changeux J. P. Detection of the nicotinic acetylcholine receptor alpha-subunit mRNA by in situ hybridization at neuromuscular junctions of 15-day-old chick striated muscles. EMBO J. 1988 Mar;7(3):603–609. doi: 10.1002/j.1460-2075.1988.tb02853.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gergen J. P., Butler B. A. Isolation of the Drosophila segmentation gene runt and analysis of its expression during embryogenesis. Genes Dev. 1988 Sep;2(9):1179–1193. doi: 10.1101/gad.2.9.1179. [DOI] [PubMed] [Google Scholar]
  13. Giulian D., Johnson B., Krebs J. F., Tapscott M. J., Honda S. A growth factor from neuronal cell lines stimulates myelin protein synthesis in mammalian brain. J Neurosci. 1991 Feb;11(2):327–336. doi: 10.1523/JNEUROSCI.11-02-00327.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Golumbeski G. S., Bardsley A., Tax F., Boswell R. E. tudor, a posterior-group gene of Drosophila melanogaster, encodes a novel protein and an mRNA localized during mid-oogenesis. Genes Dev. 1991 Nov;5(11):2060–2070. doi: 10.1101/gad.5.11.2060. [DOI] [PubMed] [Google Scholar]
  15. Holmes E., Hermanson G., Cole R., de Vellis J. Developmental expression of glial-specific mRNAs in primary cultures of rat brain visualized by in situ hybridization. J Neurosci Res. 1988 Apr;19(4):389-96, 458-65. doi: 10.1002/jnr.490190402. [DOI] [PubMed] [Google Scholar]
  16. Kachar B., Behar T., Dubois-Dalcq M. Cell shape and motility of oligodendrocytes cultured without neurons. Cell Tissue Res. 1986;244(1):27–38. doi: 10.1007/BF00218378. [DOI] [PubMed] [Google Scholar]
  17. Kim-Ha J., Smith J. L., Macdonald P. M. oskar mRNA is localized to the posterior pole of the Drosophila oocyte. Cell. 1991 Jul 12;66(1):23–35. doi: 10.1016/0092-8674(91)90136-m. [DOI] [PubMed] [Google Scholar]
  18. Kimhi Y., Palfrey C., Spector I., Barak Y., Littauer U. Z. Maturation of neuroblastoma cells in the presence of dimethylsulfoxide. Proc Natl Acad Sci U S A. 1976 Feb;73(2):462–466. doi: 10.1073/pnas.73.2.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kleiman R., Banker G., Steward O. Differential subcellular localization of particular mRNAs in hippocampal neurons in culture. Neuron. 1990 Dec;5(6):821–830. doi: 10.1016/0896-6273(90)90341-c. [DOI] [PubMed] [Google Scholar]
  20. Kristensson K., Holmes K. V., Duchala C. S., Zeller N. K., Lazzarini R. A., Dubois-Dalcq M. Increased levels of myelin basic protein transcripts in virus-induced demyelination. Nature. 1986 Aug 7;322(6079):544–547. doi: 10.1038/322544a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lawrence J. B., Singer R. H. Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell. 1986 May 9;45(3):407–415. doi: 10.1016/0092-8674(86)90326-0. [DOI] [PubMed] [Google Scholar]
  22. Macdonald P. M., Ingham P., Struhl G. Isolation, structure, and expression of even-skipped: a second pair-rule gene of Drosophila containing a homeo box. Cell. 1986 Dec 5;47(5):721–734. doi: 10.1016/0092-8674(86)90515-5. [DOI] [PubMed] [Google Scholar]
  23. Mohr E., Fehr S., Richter D. Axonal transport of neuropeptide encoding mRNAs within the hypothalamo-hypophyseal tract of rats. EMBO J. 1991 Sep;10(9):2419–2424. doi: 10.1002/j.1460-2075.1991.tb07781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Morgan F., Barbarese E., Carson J. H. Visualizing cells in three dimensions using confocal microscopy, image reconstruction and isosurface rendering: application to glial cells in mouse central nervous system. Scanning Microsc. 1992 Jun;6(2):345–357. [PubMed] [Google Scholar]
  25. Okabe S., Hirokawa N. Axonal transport. Curr Opin Cell Biol. 1989 Feb;1(1):91–97. doi: 10.1016/s0955-0674(89)80043-2. [DOI] [PubMed] [Google Scholar]
  26. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  27. Rebagliati M. R., Weeks D. L., Harvey R. P., Melton D. A. Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell. 1985 Oct;42(3):769–777. doi: 10.1016/0092-8674(85)90273-9. [DOI] [PubMed] [Google Scholar]
  28. Roach A., Boylan K., Horvath S., Prusiner S. B., Hood L. E. Characterization of cloned cDNA representing rat myelin basic protein: absence of expression in brain of shiverer mutant mice. Cell. 1983 Oct;34(3):799–806. doi: 10.1016/0092-8674(83)90536-6. [DOI] [PubMed] [Google Scholar]
  29. Shiota C., Miura M., Mikoshiba K. Developmental profile and differential localization of mRNAs of myelin proteins (MBP and PLP) in oligodendrocytes in the brain and in culture. Brain Res Dev Brain Res. 1989 Jan 1;45(1):83–94. doi: 10.1016/0165-3806(89)90010-2. [DOI] [PubMed] [Google Scholar]
  30. Singer R. H., Langevin G. L., Lawrence J. B. Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization. J Cell Biol. 1989 Jun;108(6):2343–2353. doi: 10.1083/jcb.108.6.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sundell C. L., Singer R. H. Requirement of microfilaments in sorting of actin messenger RNA. Science. 1991 Sep 13;253(5025):1275–1277. doi: 10.1126/science.1891715. [DOI] [PubMed] [Google Scholar]
  32. Taneja K. L., Lifshitz L. M., Fay F. S., Singer R. H. Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy. J Cell Biol. 1992 Dec;119(5):1245–1260. doi: 10.1083/jcb.119.5.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vallee R. B., Bloom G. S. Mechanisms of fast and slow axonal transport. Annu Rev Neurosci. 1991;14:59–92. doi: 10.1146/annurev.ne.14.030191.000423. [DOI] [PubMed] [Google Scholar]
  34. Verity A. N., Campagnoni A. T. Regional expression of myelin protein genes in the developing mouse brain: in situ hybridization studies. J Neurosci Res. 1988 Oct-Dec;21(2-4):238–248. doi: 10.1002/jnr.490210216. [DOI] [PubMed] [Google Scholar]
  35. Wang C., Lehmann R. Nanos is the localized posterior determinant in Drosophila. Cell. 1991 Aug 23;66(4):637–647. doi: 10.1016/0092-8674(91)90110-k. [DOI] [PubMed] [Google Scholar]
  36. Weir M. P., Kornberg T. Patterns of engrailed and fushi tarazu transcripts reveal novel intermediate stages in Drosophila segmentation. Nature. 1985 Dec 5;318(6045):433–439. doi: 10.1038/318433a0. [DOI] [PubMed] [Google Scholar]
  37. Wells B. D., Cantor C. R. A strong ethidium binding site in the acceptor stem of most or all transfer RNAs. Nucleic Acids Res. 1977;4(5):1667–1680. doi: 10.1093/nar/4.5.1667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wells B. D., Cantor C. R. Ribosome binding by tRNAs with fluorescent labeled 3' termini. Nucleic Acids Res. 1980 Jul 25;8(14):3229–3246. doi: 10.1093/nar/8.14.3229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yisraeli J. K., Sokol S., Melton D. A. A two-step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA. Development. 1990 Feb;108(2):289–298. doi: 10.1242/dev.108.2.289. [DOI] [PubMed] [Google Scholar]

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