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Analysis of Subcellularly Localized mRNAs Using in Situ Hybridization, mRNA Amplification, and Expression Profiling

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Abstract

Targeting of mRNAs to distinct subcellular regions occurs in all polarized cells. The mechanisms by which RNA transport occurs are poorly understood. With the advent of RNA amplification methodologies and expression profiling it is now possible to catalogue the RNAs that are targeted to particular subcellular regions. In particular, neurons are polarized cells in which dendrites receive signals from presynaptic neurons. Upon stimulation (information receipt) the dendrite processes the information such that an immediate dendritic response is generated as well as a longer-term somatic response. The integrated cellular response results in a signal that can be propagated through the axon to the next post-synaptic neuron. Much previous work has shown that mRNAs can be localized in dendrites and that local translation in dendrites can occur. In this chapter the methods for analysis of RNAs that are localized to dendrites are reviewed and a partial list of dendritically localized RNAs is presented. This information may be useful in identifying RNA regulatory regions that are responsible for specifying rate of RNA transport and the dendritic sites at which targeted RNAs dock so that they can be translated.

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REFERENCES

  1. Tiedge, H. and Brosius, J. 1996. Translational machinery in dendrites of hippocampal neurons in culture. J. Neurosci. 16(22):7171-7181.

    Google Scholar 

  2. Steward, O. and Levy, W. B. 1982. Preferential localization of polyribosomes under the base of dendritic spines in granule cells of the dentale gyrus. J. Neurosci. 2(3):284-291.

    Google Scholar 

  3. Kiss, J. 1977. Synthesis and transport of newly formed proteins in dendrites of rat hippocampal pyramid cells. An electron microscope autoradiographic study. Brain Res. 124(2):237-250.

    Google Scholar 

  4. Weiler, I. J., Wang X., and Greenough, W. T. 1994. Synapse-activated protein synthesis as a possible mechanism of plastic neural change. Prog. Brain 100:189-194.

    Google Scholar 

  5. Crino, P. B. and Eberwine, J. 1996. Molecular characterization of the dendritic growth cone: regulated mRNA transport and local protein synthesis. Neuron 17(6):1173-1187.

    Google Scholar 

  6. Eberwine, J., Miyashiro, K., Kacharmina, J. E., and Job, C. 2001. Local translation of classes of mRNAs that are targeted to neuronal dendrites. Proc. Natl. Acad. Sci. USA 98(13):7080-7085.

    Google Scholar 

  7. Job, C. and Eberwine, J. 2001. Localization and translation of mRNA in dendrites and axons. Nat. Rev. Neurosci. 2(12):889-898.

    Google Scholar 

  8. Job, C. and Eberwine, J. 2001. Identification of sites for exponential translation in living dendrites. Proc. Natl. Acad. Sci. USA 98(23):13037-13042.

    Google Scholar 

  9. Kang, H. and Schuman, E. M. 1996. A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity. Science 273(5280):1402-1406.

    Google Scholar 

  10. Huber, K. M., Kayser, M. S., and Bear, M. F. 2000. Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression. Science 288(5469):1254-1257.

    Google Scholar 

  11. Spencer, C. M. and Eberwine, J. 1999. Cytoplasmic proteins interact with a translational control element in the protein-coding region of proopiomelanocortin mRNA. DNA Cell Biol. 18(1):39-49.

    Google Scholar 

  12. Eberwine, J. H., Spencer, C., Newell, D., and Hoffman, A. R. 1993. mRNA structure, in situ, as assessed by microscopic techniques. Microsc. Res. Tech. 25(1):19-28.

    Google Scholar 

  13. Roberts, J. L., Eberwine, J. H., and Gee, C. E. 1983. Analysis of POMC gene expression by transcription assay and in situ hybridization histochemistry. Cold Spring Harb. Symp. Quant. Biol. 48 Pt. 1:385-391.

    Google Scholar 

  14. Tecott, L. H., Barchas, J. D., and Eberwine, J. H. 1988. In situ transcription: specific synthesis of complementary DNA in fixed tissue sections. Science 240(4859):1661-1664.

    Google Scholar 

  15. Saiki, R. K., Bugawan, T. L., Horn, G. T., Mullis, K. B., and Erlich, H. A. 1986. Analysis of enzymatically amplified betaglobin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 324(6093):163-166.

    Google Scholar 

  16. Hagen-Mann, K. and Mann, W. 1995. RT-PCR and alternative methods to PCR for in vitro amplification of nucleic acids. Exp. Clin. Endocrinol. Diabetes 103(3):150-155.

    Google Scholar 

  17. Eberwine, J., Yeh, H., Miyashiro, K., Cao, Y., Nair, S., Finnell, R., Zettel, M., and Coleman, P. 1992. Analysis of gene expression in single live neurons. Proc. Natl. Acad. Sci. USA 89(7):3010-3014.

    Google Scholar 

  18. Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., and Eberwine, J. H. 1990. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA 87(5):1663-1667.

    Google Scholar 

  19. Luo, L., Salunga, R. C., Guo, H., Bittner, A., Joy, K. C., Galindo, J. E., Xiao, H., Rogers, K. E., Wan, J. S., Jackson, M. R., and Erlander, M. G. 1999. Gene expression profiles of laser-captured adjacent neuronal subtypes. Nat. Med. 5(1):117-122.

    Google Scholar 

  20. Zarrinkar, P. P., Mainquist, J. K., Zamora, M., Stern, D., Welsh, J. B., Sapinoso, L. M., Hampton, G. M., and Lockhart, D. J. 2001. Arrays of arrays for high-throughput gene expression profiling. Genome Res. 11(7):1256-1261.

    Google Scholar 

  21. Miyashiro, K., Dichter, M., and Eberwine, J. 1994. On the nature and differential distribution of mRNAs in hippocampal neurites: implications for neuronal functioning. Proc. Natl. Acad. Sci. USA 91(23):10800-10804.

    Google Scholar 

  22. Steward, O., Wallace, C. S., Lyford, G. L., and Worley, P. F. 1998. Synaptic activation causes the mRNA for the IEG Arc to localize selectively near activated postsynaptic sites on dendrites. Neuron 21(4):741-751.

    Google Scholar 

  23. Steward, O. and Falk, P. M. 1985. Polyribosomes under developing spine synapses: growth specializations of dendrites at sites of synaptogenesis. J. Neurosci. Res. 13(1-2):75-88.

    Google Scholar 

  24. Torre, E. R. and Steward, O. 1996. Protein synthesis within dendrites: glycosylation of newly synthesized proteins in dendrites of hippocampal neurons in culture. J. Neurosci. 16(19):5967-5978.

    Google Scholar 

  25. Kacharmina, J. E., Job, C., Crino, P., and Eberwine, J. 2000. Stimulation of glutamate receptor protein synthesis and membrane insertion within isolated neuronal dendrites. Proc. Natl. Acad. Sci. USA 97(21):11545-11550.

    Google Scholar 

  26. Meshorer, E., et al. 2002. Alternative splicing and neuritic mRNA translocation under long-term neuronal hypersensitivity. Science 295(5554):508-512.

    Google Scholar 

  27. Kleiman, R., Banker, G., and Steward, O. 1994. Development of subcellular mRNA compartmentation in hippocampal neurons in culture. J. Neurosci. 14(3 Pt 1):1130-1140

    Google Scholar 

  28. Laurent-Demir, C., et al. 2000. Differential regulation of Ca(2+)-calmodulin stimulated and Ca(2+)-insensitive adenylyl cyclase messenger RNA in intact and denervated mouse hippocampus. Neuroscience 96(2):267-274.

    Google Scholar 

  29. Strong, M. J., et al. 1990. The temporal expression of amyloid precursor protein mRNA in vitro in dissociated hippocampal neuron cultures. Exp. Neurol. 109(2):171-179.

    Google Scholar 

  30. Lyford, G. L., et al. 1995. Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites. Neuron. 14(2):433-445.

    Google Scholar 

  31. Tongiorgi, E., Righi, M., and Cattaneo, A. 1997. Activity-dependent dendritic targeting of BDNF and TrkB mRNAs in hippocampal neurons. J. Neurosci. 17(24):9492-9505.

    Google Scholar 

  32. Berry, F. B., and Brown, I. R. 1996. CaMI mRNA is localized to apical dendrites during postnatal development of neurons in the rat brain. J. Neurosci. Res. 43(5):565-575.

    Google Scholar 

  33. Burgin, K. E., et al. 1990. In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain. J. Neurosci. 10(6):1788-1798.

    Google Scholar 

  34. Eastwood, S. L. and Harrison, P. J. 2000. Complexin I and II mRNAs in the human hippocampus. Mol. Psychiatry 5(4):341.

    Google Scholar 

  35. Hevner, R. F., and Wong-Riley, M. T. 1991. Neuronal expression of nuclear and mitochondrial genes for cytochrome oxidase (CO) subunits analyzed by in situ hybridization: comparison with CO activity and protein. J. Neurosci. 11(7):1942-1958.

    Google Scholar 

  36. Neuner-Jehle, M., et al. 1996. Characterization and sleep deprivation-induced expression modulation of dendrin, a novel dendritic protein in rat brain neurons. J. Neurosci. Res. 46(2):138-151.

    Google Scholar 

  37. Herb, A., et al. 1997. Prominent dendritic localization in forebrain neurons of a novel mRNA and its product, dendrin. Mol. Cell Neurosci. 8(5):367-374.

    Google Scholar 

  38. Ishimoto, T., et al. 2000. Dendritic translocation of the rat ferritin H chain mRNA. Biochem. Biophys. Res. Commun. 272(3):789-793.

    Google Scholar 

  39. Sperk, G., et al. 1998. Expression of GABA(A) receptor subunits in the hippocampus of the rat after kainic acid-induced seizures. Epilepsy Res. 32(1-2):129-139.

    Google Scholar 

  40. Chicurel, M. E., Terrian, D. M., and Potter, H. 1993. mRNA at the synapse: analysis of a synaptosomal preparation enriched in hippocampal dendritic spines. J. Neurosci. 13(9):4054-4063.

    Google Scholar 

  41. Landry, C. F., et al. 1994. Cellular influences on RNA sorting in neurons and glia: an in situ hybridization histochemical study. Brain Res. Mol. Brain Res. 27(1):1-11.

    Google Scholar 

  42. Racca, C., Gardiol, A., and Triller, A. 1997. Dendritic and postsynaptic localizations of glycine receptor alpha subunit mRNAs. J. Neurosci. 17(5):1691-1700.

    Google Scholar 

  43. Watson, J. B., et al. 1994. G-protein gamma 7 subunit is selectively expressed in medium-sized neurons and dendrites of the rat neostriatum. J. Neurosci. Res. 39(1):108-116.

    Google Scholar 

  44. Link, W., et al. 1995. Somatodendritic expression of an immediate early gene is regulated by synaptic activity. Proc. Natl. Acad. Sci. USA 92(12):5734-5738.

    Google Scholar 

  45. Foster, J. A., and Brown, I. R. 1996. Intracellular localization of heat shock mRNAs (hsc70 and hsp70) to neural cell bodies and processes in the control and hyperthermic rabbit brain. J. Neurosci. Res. 46(6):652-665.

    Google Scholar 

  46. Furuichi, T., et al. 1993. Widespread expression of inositol 1,4,5-triphosphate receptor type 1 gene (Insp3r1) in the mouse central nervous system. Receptors Channels 1(1):11-24.

    Google Scholar 

  47. Bloch, B., et al. 1990. Presence of neuropeptide messenger RNAs in neuronal processes. Neurosci. Lett. 109(3):259-264.

    Google Scholar 

  48. Severt, W. L., et al. 1999. The suppression of testis-brain RNA binding protein and kinesin heavy chain disrupts mRNA sorting in dendrites. J. Cell. Sci. 112(Pt 21):3691-3702.

    Google Scholar 

  49. Garner, C. C., Tucker, R. P., and Matus, A. 1988. Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites. Nature 336(6200):674-677.

    Google Scholar 

  50. Grant, A. L., et al. 1996. Characterization of the rat hippocalcin gene: the 5´ flanking region directs expression to the hippocampus. Neuroscience 75(4):1099-1115.

    Google Scholar 

  51. Matsuoka, Y., et al. 2002. A chromodomain-containing nuclear protein, MRG15 is expressed as a novel type of dendritic mRNA in neurons. Neurosci. Res. 42(4):299-308.

    Google Scholar 

  52. Paradies, M. A., and Steward, O. 1997. Multiple subcellular mRNA distribution patterns in neurons: a nonisotopic in situ hybridization analysis. J. Neurobiol. 33(4):473-493.

    Google Scholar 

  53. Krishna, N. S., et al. 1995. Human olfactory receptor neurons contain OMP mRNA in their dendritic and axonal processes. Neuroreport 6(5):817-821.

    Google Scholar 

  54. Bian, F., et al. 1996. Differential mRNA transport and the regulation of protein synthesis: selective sensitivity of Purkinje cell dendritic mRNAs to translational inhibition. Mol. Cell Neurosci. 7(2):116-133.

    Google Scholar 

  55. Moriya, M., and Tanaka, S. 1994. Prominent expression of protein kinase C (gamma) mRNA in the dendrite-rich neuropil of mice cerebellum at the critical period for synaptogenesis. Neuroreport. 5(8):929-932.

    Google Scholar 

  56. Gelot, A., et al. 1996. Alpha-brain spectrin mRNA belongs to the population of intradendritically transported mRNAs. Neuroreport. 8(1):113-116.

    Google Scholar 

  57. Kosik, K. S., et al. 1989. Tau in situ hybridization in normal and Alzheimer brain: localization in the somatodendritic compartment. Ann. Neurol. 26(3):352-361.

    Google Scholar 

  58. Dumas, S., et al. 1990. Tyrosine hydroxylase gene expression in human ventral mesencephalon: detection of tyrosine hydroxylase messenger RNA in neurites. J. Neurosci. Res. 25(4):569-575.

    Google Scholar 

  59. Mohr, E., Morris, J. F., and Richter, D. 1995. Differential subcellular mRNA targeting: deletion of a single nucleotide prevents the transport to axons but not to dendrites of rat hypothalamic magnocellular neurons. Proc. Natl. Acad. Sci. USA 92(10):4377-4381.

    Google Scholar 

  60. Eberwine, J., et al. 2001. Transcription factors in dendrites: dendritic imprinting of the cellular nucleus. Results Probl. Cell Differ. 34:57-68.

    Google Scholar 

  61. Miyashiro, K. and Eberwine, J. unpublished observations.

  62. Bodian, D. 1965. A suggestive relationship of nerve cell RNA with specific synaptic sites. Proc. Natl. Acad. Sci. USA 53:418-425.

    Google Scholar 

  63. Miyashiro, K., Finnel, R. H., and Eberwine, J. Map kinase pathways regulate ank mRNA localization to neuronal dendrites. Submitted.

  64. Therianos, S., et al. Transcription factor mRNA and protein in dendrites: Interneuronal transport of Nkx-2.2. Submitted.

  65. Miyashiro, K., et al. RNA cargoes associating with FMRP reveal deficits in cellular frunctionin in FMR1 null mice. Submitted.

  66. Crino, P., et al. 1998. Presence and phosphorylation of transcription factors in dendrites. Proc. Natl. Acad. Sci. USA 95:2313-2318.

    Google Scholar 

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Eberwine, J., Belt, B., Kacharmina, J.E. et al. Analysis of Subcellularly Localized mRNAs Using in Situ Hybridization, mRNA Amplification, and Expression Profiling. Neurochem Res 27, 1065–1077 (2002). https://doi.org/10.1023/A:1020956805307

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