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J Cell Biol. 1994 August 2; 126(4): 863–876.
PMCID: PMC2120111

Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts


Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize "individual" poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (< 10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and alpha-actinin, and the elongation factor, EF-1 alpha (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a "microdomain" where mRNA is attached and functionally expressed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ainger K, Avossa D, Morgan F, Hill SJ, Barry C, Barbarese E, Carson JH. Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes. J Cell Biol. 1993 Oct;123(2):431–441. [PMC free article] [PubMed]
  • Altman LG, Schneider BG, Papermaster DS. Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy. J Histochem Cytochem. 1984 Nov;32(11):1217–1223. [PubMed]
  • Bassell GJ. High resolution distribution of mRNA within the cytoskeleton. J Cell Biochem. 1993 Jun;52(2):127–133. [PubMed]
  • Bassell GJ, Singer RH, Kosik KS. Association of poly(A) mRNA with microtubules in cultured neurons. Neuron. 1994 Mar;12(3):571–582. [PubMed]
  • Ben-Ze'ev A, Horowitz M, Skolnik H, Abulafia R, Laub O, Aloni Y. The metabolism of SV40 RNA is associated with the cytoskeletal framework. Virology. 1981 Jun;111(2):475–487. [PubMed]
  • Bendayan M. Ultrastructural localization of actin in muscle, epithelial and secretory cells by applying the protein A-gold immunocytochemical technique. Histochem J. 1983 Jan;15(1):39–58. [PubMed]
  • Biggiogera M, Fakan S, Kaufmann SH, Black A, Shaper JH, Busch H. Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus. J Histochem Cytochem. 1989 Sep;37(9):1371–1374. [PubMed]
  • Bonneau AM, Darveau A, Sonenberg N. Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure. J Cell Biol. 1985 Apr;100(4):1209–1218. [PMC free article] [PubMed]
  • Brawerman G. The Role of the poly(A) sequence in mammalian messenger RNA. CRC Crit Rev Biochem. 1981;10(1):1–38. [PubMed]
  • Carter KC, Taneja KL, Lawrence JB. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol. 1991 Dec;115(5):1191–1202. [PMC free article] [PubMed]
  • Cervera M, Dreyfuss G, Penman S. Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells. Cell. 1981 Jan;23(1):113–120. [PubMed]
  • Christensen AK, Kahn LE, Bourne CM. Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary. Am J Anat. 1987 Jan;178(1):1–10. [PubMed]
  • Eberwine J, Spencer C, Miyashiro K, Mackler S, Finnell R. Complementary DNA synthesis in situ: methods and applications. Methods Enzymol. 1992;216:80–100. [PubMed]
  • Erickson PA, Feinstein SC, Lewis GP, Fisher SK. Glial fibrillary acidic protein and its mRNA: ultrastructural detection and determination of changes after CNS injury. J Struct Biol. 1992 Mar-Apr;108(2):148–161. [PubMed]
  • Farmer SR, Ben-Ze'av A, Benecke BJ, Penman S. Altered translatability of messenger RNA from suspended anchorage-dependent fibroblasts: reversal upon cell attachment to a surface. Cell. 1978 Oct;15(2):627–637. [PubMed]
  • Fulton AB, Wan KM, Penman S. The spatial distribution of polyribosomes in 3T3 cells and the associated assembly of proteins into the skeletal framework. Cell. 1980 Jul;20(3):849–857. [PubMed]
  • Hartwig JH, Kwiatkowski DJ. Actin-binding proteins. Curr Opin Cell Biol. 1991 Feb;3(1):87–97. [PubMed]
  • Hesketh JE, Pryme IF. Interaction between mRNA, ribosomes and the cytoskeleton. Biochem J. 1991 Jul 1;277(Pt 1):1–10. [PubMed]
  • Hsu MT, Coca-Prados M. Electron microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells. Nature. 1979 Jul 26;280(5720):339–340. [PubMed]
  • Jeffery WR. Messenger RNA in the cytoskeletal framework: analysis by in situ hybridization. J Cell Biol. 1982 Oct;95(1):1–7. [PMC free article] [PubMed]
  • Kislauskis EH, Li Z, Singer RH, Taneja KL. Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J Cell Biol. 1993 Oct;123(1):165–172. [PMC free article] [PubMed]
  • Ladhoff AM, Uerlings I, Rosenthal S. Electron microscopic evidence of circular molecules in 9-S globin mRNA from rabbit reticulocytes. Mol Biol Rep. 1981 May 22;7(1-3):101–106. [PubMed]
  • Lenk R, Penman S. The cytoskeletal framework and poliovirus metabolism. Cell. 1979 Feb;16(2):289–301. [PubMed]
  • Lenk R, Ransom L, Kaufmann Y, Penman S. A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell. 1977 Jan;10(1):67–78. [PubMed]
  • Munroe D, Jacobson A. Tales of poly(A): a review. Gene. 1990 Jul 16;91(2):151–158. [PubMed]
  • Nakayasu H, Ueda K. Ultrastructural localization of actin in nuclear matrices from mouse leukemia L5178Y cells. Cell Struct Funct. 1985 Sep;10(3):305–309. [PubMed]
  • Nakayasu H, Ueda K. Association of rapidly-labelled RNAs with actin in nuclear matrix from mouse L5178Y cells. Exp Cell Res. 1985 Oct;160(2):319–330. [PubMed]
  • Nakayasu H, Ueda K. Preferential association of acidic actin with nuclei and nuclear matrix from mouse leukemia L5178Y cells. Exp Cell Res. 1986 Apr;163(2):327–336. [PubMed]
  • Nickerson JA, Penman S. Bio Vision: microscopy in three dimensions. Semin Cell Biol. 1991 Apr;2(2):117–129. [PubMed]
  • Nielsen P, Goelz S, Trachsel H. The role of the cytoskeleton in eukaryotic protein synthesis. (A minireview). Cell Biol Int Rep. 1983 Apr;7(4):245–254. [PubMed]
  • Ornelles DA, Fey EG, Penman S. Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis. Mol Cell Biol. 1986 May;6(5):1650–1662. [PMC free article] [PubMed]
  • Pokrywka NJ, Stephenson EC. Microtubules mediate the localization of bicoid RNA during Drosophila oogenesis. Development. 1991 Sep;113(1):55–66. [PubMed]
  • Pondel MD, King ML. Localized maternal mRNA related to transforming growth factor beta mRNA is concentrated in a cytokeratin-enriched fraction from Xenopus oocytes. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7612–7616. [PubMed]
  • Pudney J, Singer RH. Electron microscopic visualization of the filamentous reticulum in whole cultured presumptive chick myoblasts. Am J Anat. 1979 Nov;156(3):321–336. [PubMed]
  • Sachs AB, Davis RW. The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation. Cell. 1989 Sep 8;58(5):857–867. [PubMed]
  • Scheer U, Hinssen H, Franke WW, Jockusch BM. Microinjection of actin-binding proteins and actin antibodies demonstrates involvement of nuclear actin in transcription of lampbrush chromosomes. Cell. 1984 Nov;39(1):111–122. [PubMed]
  • Sheiness D, Darnell JE. Polyadenylic acid segment in mRNA becomes shorter with age. Nat New Biol. 1973 Feb 28;241(113):265–268. [PubMed]
  • Singer RH. The cytoskeleton and mRNA localization. Curr Opin Cell Biol. 1992 Feb;4(1):15–19. [PubMed]
  • Singer RH. RNA zipcodes for cytoplasmic addresses. Curr Biol. 1993 Oct 1;3(10):719–721. [PubMed]
  • Sundell CL, Singer RH. Requirement of microfilaments in sorting of actin messenger RNA. Science. 1991 Sep 13;253(5025):1275–1277. [PubMed]
  • Singer RH, Langevin GL, Lawrence JB. Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization. J Cell Biol. 1989 Jun;108(6):2343–2353. [PMC free article] [PubMed]
  • Taneja KL, Lifshitz LM, Fay FS, Singer RH. 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. [PMC free article] [PubMed]
  • Tecott LH, Barchas JD, Eberwine JH. In situ transcription: specific synthesis of complementary DNA in fixed tissue sections. Science. 1988 Jun 17;240(4859):1661–1664. [PubMed]
  • van Venrooij WJ, Sillekens PT, van Eekelen CA, Reinders RJ. On the association of mRNA with the cytoskeleton in uninfected and adenovirus-infected human KB cells. Exp Cell Res. 1981 Sep;135(1):79–91. [PubMed]
  • Vandekerckhove J. Actin-binding proteins. Curr Opin Cell Biol. 1990 Feb;2(1):41–50. [PubMed]
  • Visa N, Puvion-Dutilleul F, Harper F, Bachellerie JP, Puvion E. Intranuclear distribution of poly(A) RNA determined by electron microscope in situ hybridization. Exp Cell Res. 1993 Sep;208(1):19–34. [PubMed]
  • Yang F, Demma M, Warren V, Dharmawardhane S, Condeelis J. Identification of an actin-binding protein from Dictyostelium as elongation factor 1a. Nature. 1990 Oct 4;347(6292):494–496. [PubMed]
  • Zambetti G, Schmidt W, Stein G, Stein J. Subcellular localization of histone messenger RNAs on cytoskeleton-associated free polysomes in HeLa S3 cells. J Cell Physiol. 1985 Nov;125(2):345–353. [PubMed]

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