• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. Aug 2, 1994; 126(4): 877–899.
PMCID: PMC2120126

In vivo analysis of the stability and transport of nuclear poly(A)+ RNA

Abstract

We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre- mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.

Full Text

The Full Text of this article is available as a PDF (13M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Akey CW, Goldfarb DS. Protein import through the nuclear pore complex is a multistep process. J Cell Biol. 1989 Sep;109(3):971–982. [PMC free article] [PubMed]
  • Andrade LE, Tan EM, Chan EK. Immunocytochemical analysis of the coiled body in the cell cycle and during cell proliferation. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1947–1951. [PMC free article] [PubMed]
  • Belgrader P, Siegel AJ, Berezney R. A comprehensive study on the isolation and characterization of the HeLa S3 nuclear matrix. J Cell Sci. 1991 Mar;98(Pt 3):281–291. [PubMed]
  • Berezney R, Coffey DS. Identification of a nuclear protein matrix. Biochem Biophys Res Commun. 1974 Oct 23;60(4):1410–1417. [PubMed]
  • Berezney R. The nuclear matrix: a heuristic model for investigating genomic organization and function in the cell nucleus. J Cell Biochem. 1991 Oct;47(2):109–123. [PubMed]
  • Bernhard W. A new staining procedure for electron microscopical cytology. J Ultrastruct Res. 1969 May;27(3):250–265. [PubMed]
  • Blobel G. Gene gating: a hypothesis. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8527–8529. [PMC free article] [PubMed]
  • Bouvier D, Hubert J, Sève AP, Bouteille M. Structural aspects of intranuclear matrix disintegration upon RNase digestion of HeLa cell nuclei. Eur J Cell Biol. 1985 Mar;36(2):323–333. [PubMed]
  • Brasch K. Drug and metabolite-induced perturbations in nuclear structure and function: a review. Biochem Cell Biol. 1990 Feb;68(2):408–426. [PubMed]
  • Brasch K, Ochs RL. Nuclear bodies (NBs): a newly "rediscovered" organelle. Exp Cell Res. 1992 Oct;202(2):211–223. [PubMed]
  • Brawerman G, Diez J. Metabolism of the polyadenylate sequence of nuclear RNA and messenger RNA in mammalian cells. Cell. 1975 Jul;5(3):271–280. [PubMed]
  • Brockdorff N, Ashworth A, Kay GF, McCabe VM, Norris DP, Cooper PJ, Swift S, Rastan S. The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell. 1992 Oct 30;71(3):515–526. [PubMed]
  • Brown CJ, Willard HF. The human X-inactivation centre is not required for maintenance of X-chromosome inactivation. Nature. 1994 Mar 10;368(6467):154–156. [PubMed]
  • Brown CJ, Hendrich BD, Rupert JL, Lafrenière RG, Xing Y, Lawrence J, Willard HF. The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus. Cell. 1992 Oct 30;71(3):527–542. [PubMed]
  • Carmo-Fonseca M, Pepperkok R, Sproat BS, Ansorge W, Swanson MS, Lamond AI. In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells. EMBO J. 1991 Jul;10(7):1863–1873. [PMC free article] [PubMed]
  • Carmo-Fonseca M, Tollervey D, Pepperkok R, Barabino SM, Merdes A, Brunner C, Zamore PD, Green MR, Hurt E, Lamond AI. Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery. EMBO J. 1991 Jan;10(1):195–206. [PMC free article] [PubMed]
  • Carmo-Fonseca M, Pepperkok R, Carvalho MT, Lamond AI. Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 snRNPs in coiled bodies. J Cell Biol. 1992 Apr;117(1):1–14. [PMC free article] [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]
  • Carter KC, Bowman D, Carrington W, Fogarty K, McNeil JA, Fay FS, Lawrence JB. A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. Science. 1993 Feb 26;259(5099):1330–1335. [PubMed]
  • Chan VL, Whitmore GF, Siminovitch L. Mammalian cells with altered forms of RNA polymerase II. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3119–3123. [PMC free article] [PubMed]
  • Chan EK, Sullivan KF, Tan EM. Ribonucleoprotein SS-B/La belongs to a protein family with consensus sequences for RNA-binding. Nucleic Acids Res. 1989 Mar 25;17(6):2233–2244. [PMC free article] [PubMed]
  • Ciejek EM, Nordstrom JL, Tsai MJ, O'Malley BW. Ribonucleic acid precursors are associated with the chick oviduct nuclear matrix. Biochemistry. 1982 Sep 28;21(20):4945–4953. [PubMed]
  • Cordes VC, Reidenbach S, Köhler A, Stuurman N, van Driel R, Franke WW. Intranuclear filaments containing a nuclear pore complex protein. J Cell Biol. 1993 Dec;123(6 Pt 1):1333–1344. [PMC free article] [PubMed]
  • Dargemont C, Kühn LC. Export of mRNA from microinjected nuclei of Xenopus laevis oocytes. J Cell Biol. 1992 Jul;118(1):1–9. [PMC free article] [PubMed]
  • Deerinck TJ, Martone ME, Lev-Ram V, Green DP, Tsien RY, Spector DL, Huang S, Ellisman MH. Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy. J Cell Biol. 1994 Aug;126(4):901–910. [PMC free article] [PubMed]
  • Dworetzky SI, Feldherr CM. Translocation of RNA-coated gold particles through the nuclear pores of oocytes. J Cell Biol. 1988 Mar;106(3):575–584. [PMC free article] [PubMed]
  • Eckner R, Ellmeier W, Birnstiel ML. Mature mRNA 3' end formation stimulates RNA export from the nucleus. EMBO J. 1991 Nov;10(11):3513–3522. [PMC free article] [PubMed]
  • Fakan S, Bernhard W. Nuclear labelling after prolonged 3H-uridine incorporation as visualized by high resolution autoradiography. Exp Cell Res. 1973 Jun;79(2):431–444. [PubMed]
  • Fakan S, Puvion E. The ultrastructural visualization of nucleolar and extranucleolar RNA synthesis and distribution. Int Rev Cytol. 1980;65:255–299. [PubMed]
  • Fakan S, Leser G, Martin TE. Ultrastructural distribution of nuclear ribonucleoproteins as visualized by immunocytochemistry on thin sections. J Cell Biol. 1984 Jan;98(1):358–363. [PMC free article] [PubMed]
  • Feldherr CM, Akin D. EM visualization of nucleocytoplasmic transport processes. Electron Microsc Rev. 1990;3(1):73–86. [PubMed]
  • Fey EG, Krochmalnic G, Penman S. The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy. J Cell Biol. 1986 May;102(5):1654–1665. [PMC free article] [PubMed]
  • Fu XD, Maniatis T. Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature. 1990 Feb 1;343(6257):437–441. [PubMed]
  • Goldberg MW, Allen TD. High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores. J Cell Biol. 1992 Dec;119(6):1429–1440. [PMC free article] [PubMed]
  • Green MR. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. [PubMed]
  • Hamm J, Mattaj IW. Monomethylated cap structures facilitate RNA export from the nucleus. Cell. 1990 Oct 5;63(1):109–118. [PubMed]
  • He DC, Nickerson JA, Penman S. Core filaments of the nuclear matrix. J Cell Biol. 1990 Mar;110(3):569–580. [PMC free article] [PubMed]
  • Herman RC, Williams JG, Penman S. Message and non-message sequences adjacent to poly(A) in steady state heterogeneous nuclear RNA of HeLa cells. Cell. 1976 Mar;7(3):429–437. [PubMed]
  • Hinshaw JE, Carragher BO, Milligan RA. Architecture and design of the nuclear pore complex. Cell. 1992 Jun 26;69(7):1133–1141. [PubMed]
  • Hogan NC, Traverse KL, Sullivan DE, Pardue ML. The nucleus-limited Hsr-omega-n transcript is a polyadenylated RNA with a regulated intranuclear turnover. J Cell Biol. 1994 Apr;125(1):21–30. [PMC free article] [PubMed]
  • Huang S, Spector DL. Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. Genes Dev. 1991 Dec;5(12A):2288–2302. [PubMed]
  • Huang S, Spector DL. U1 and U2 small nuclear RNAs are present in nuclear speckles. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):305–308. [PMC free article] [PubMed]
  • Ingles CJ, Guialis A, Lam J, Siminovitch L. Alpha-Amanitin resistance of RNA polymerase II in mutant Chinese hamster ovary cell lines. J Biol Chem. 1976 May 10;251(9):2729–2734. [PubMed]
  • Jackson DA, Hassan AB, Errington RJ, Cook PR. Visualization of focal sites of transcription within human nuclei. EMBO J. 1993 Mar;12(3):1059–1065. [PMC free article] [PubMed]
  • Jacob ST, Muecke W, Sajdel EM, Munro HN. Evidence for extranucleolar control of RNA synthesis in the nucleolus. Biochem Biophys Res Commun. 1970 Jul 27;40(2):334–342. [PubMed]
  • Jarnik M, Aebi U. Toward a more complete 3-D structure of the nuclear pore complex. J Struct Biol. 1991 Dec;107(3):291–308. [PubMed]
  • Kedinger C, Gniazdowski M, Mandel JL, Jr, Gissinger F, Chambon P. Alpha-amanitin: a specific inhibitor of one of two DNA-pendent RNA polymerase activities from calf thymus. Biochem Biophys Res Commun. 1970 Jan 6;38(1):165–171. [PubMed]
  • Kramerov DA, Tillib SV, Shumyatsky GP, Georgiev GP. The most abundant nascent poly(A) + RNAs are transcribed by RNA polymerase III in murine tumor cells. Nucleic Acids Res. 1990 Aug 11;18(15):4499–4506. [PMC free article] [PubMed]
  • Lamond AI, Carmo-Fonseca M. The coiled body. Trends Cell Biol. 1993 Jun;3(6):198–204. [PubMed]
  • Lindell TJ, Weinberg F, Morris PW, Roeder RG, Rutter WJ. Specific inhibition of nuclear RNA polymerase II by alpha-amanitin. Science. 1970 Oct 23;170(3956):447–449. [PubMed]
  • Long BH, Schrier WH. Isolation from Friend erythroleukemia cells of an RNase-sensitive nuclear matrix fibril fraction containing hnRNA and snRNA. Biol Cell. 1983;48(2-3):99–108. [PubMed]
  • Mariman EC, van Eekelen CA, Reinders RJ, Berns AJ, van Venrooij WJ. Adenoviral heterogeneous nuclear RNA is associated with the host nuclear matrix during splicing. J Mol Biol. 1982 Jan 5;154(1):103–119. [PubMed]
  • Martin TE, Barghusen SC, Leser GP, Spear PG. Redistribution of nuclear ribonucleoprotein antigens during herpes simplex virus infection. J Cell Biol. 1987 Nov;105(5):2069–2082. [PMC free article] [PubMed]
  • Mehlin H, Skoglund U, Daneholt B. Transport of Balbiani ring granules through nuclear pores in Chironomus tentans. Exp Cell Res. 1991 Mar;193(1):72–77. [PubMed]
  • Mehlin H, Daneholt B, Skoglund U. Translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography. Cell. 1992 May 15;69(4):605–613. [PubMed]
  • O'Keefe RT, Mayeda A, Sadowski CL, Krainer AR, Spector DL. Disruption of pre-mRNA splicing in vivo results in reorganization of splicing factors. J Cell Biol. 1994 Feb;124(3):249–260. [PMC free article] [PubMed]
  • Penman S, Rosbash M, Penman M. Messenger and heterogeneous nuclear RNA in HeLa cells: differential inhibition by cordycepin. Proc Natl Acad Sci U S A. 1970 Dec;67(4):1878–1885. [PMC free article] [PubMed]
  • Perry RP, Kelley DE, LaTorre J. Synthesis and turnover of nuclear and cytoplasmic polyadenylic acid in mouse L cells. J Mol Biol. 1974 Jan 25;82(3):315–331. [PubMed]
  • Phelan A, Carmo-Fonseca M, McLaughlan J, Lamond AI, Clements JB. A herpes simplex virus type 1 immediate-early gene product, IE63, regulates small nuclear ribonucleoprotein distribution. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9056–9060. [PMC free article] [PubMed]
  • Puvion E, Bernhard W. Ribonucleoprotein components in liver cell nuclei as visualized by cryoultramicrotomy. J Cell Biol. 1975 Oct;67(1):200–214. [PMC free article] [PubMed]
  • Raska I, Andrade LE, Ochs RL, Chan EK, Chang CM, Roos G, Tan EM. Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies. Exp Cell Res. 1991 Jul;195(1):27–37. [PubMed]
  • Reed R. Protein composition of mammalian spliceosomes assembled in vitro. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8031–8035. [PMC free article] [PubMed]
  • Schindler M, Jiang LW. Nuclear actin and myosin as control elements in nucleocytoplasmic transport. J Cell Biol. 1986 Mar;102(3):859–862. [PMC free article] [PubMed]
  • Schröder HC, Friese U, Bachmann M, Zaubitzer T, Müller WE. Energy requirement and kinetics of transport of poly(A)-free histone mRNA compared to poly(A)-rich mRNA from isolated L-cell nuclei. Eur J Biochem. 1989 Apr 15;181(1):149–158. [PubMed]
  • Smith HC, Ochs RL, Fernandez EA, Spector DL. Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: further evidence for an in situ nuclear matrix. Mol Cell Biochem. 1986 May;70(2):151–168. [PubMed]
  • Spector DL. Higher order nuclear organization: three-dimensional distribution of small nuclear ribonucleoprotein particles. Proc Natl Acad Sci U S A. 1990 Jan;87(1):147–151. [PMC free article] [PubMed]
  • Spector DL. Macromolecular domains within the cell nucleus. Annu Rev Cell Biol. 1993;9:265–315. [PubMed]
  • Spector DL, Schrier WH, Busch H. Immunoelectron microscopic localization of snRNPs. Biol Cell. 1983;49(1):1–10. [PubMed]
  • Spector DL, Fu XD, Maniatis T. Associations between distinct pre-mRNA splicing components and the cell nucleus. EMBO J. 1991 Nov;10(11):3467–3481. [PMC free article] [PubMed]
  • Spector DL, Lark G, Huang S. Differences in snRNP localization between transformed and nontransformed cells. Mol Biol Cell. 1992 May;3(5):555–569. [PMC free article] [PubMed]
  • Spector DL, O'Keefe RT, Jiménez-García LF. Dynamics of transcription and pre-mRNA splicing within the mammalian cell nucleus. Cold Spring Harb Symp Quant Biol. 1993;58:799–805. [PubMed]
  • Stirpe F, Fiume L. Studies on the pathogenesis of liver necrosis by alpha-amanitin. Effect of alpha-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei. Biochem J. 1967 Nov;105(2):779–782. [PMC free article] [PubMed]
  • Tamm I, Sehgal PB. Halobenzimidazole ribosides and RNA synthesis of cells and viruses. Adv Virus Res. 1978;22:187–258. [PubMed]
  • Thiry M. Differential location of nucleic acids within interchromatin granule clusters. Eur J Cell Biol. 1993 Dec;62(2):259–269. [PubMed]
  • Turner BM, Franchi L. Identification of protein antigens associated with the nuclear matrix and with clusters of interchromatin granules in both interphase and mitotic cells. J Cell Sci. 1987 Mar;87(Pt 2):269–282. [PubMed]
  • van Eekelen CA, van Venrooij WJ. hnRNA and its attachment to a nuclear protein matrix. J Cell Biol. 1981 Mar;88(3):554–563. [PMC free article] [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]
  • Vogelstein B, Hunt BF. A subset of small nuclear ribonucleoprotein particle antigens is a component of the nuclear matrix. Biochem Biophys Res Commun. 1982 Apr 14;105(3):1224–1232. [PubMed]
  • Wansink DG, Schul W, van der Kraan I, van Steensel B, van Driel R, de Jong L. Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J Cell Biol. 1993 Jul;122(2):283–293. [PMC free article] [PubMed]
  • Weinmann R, Roeder RG. Role of DNA-dependent RNA polymerase 3 in the transcription of the tRNA and 5S RNA genes. Proc Natl Acad Sci U S A. 1974 May;71(5):1790–1794. [PMC free article] [PubMed]
  • Xing YG, Lawrence JB. Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation. J Cell Biol. 1991 Mar;112(6):1055–1063. [PMC free article] [PubMed]
  • Xing Y, Johnson CV, Dobner PR, Lawrence JB. Higher level organization of individual gene transcription and RNA splicing. Science. 1993 Feb 26;259(5099):1326–1330. [PubMed]
  • Zachar Z, Kramer J, Mims IP, Bingham PM. Evidence for channeled diffusion of pre-mRNAs during nuclear RNA transport in metazoans. J Cell Biol. 1993 May;121(4):729–742. [PMC free article] [PubMed]
  • Zeitlin S, Parent A, Silverstein S, Efstratiadis A. Pre-mRNA splicing and the nuclear matrix. Mol Cell Biol. 1987 Jan;7(1):111–120. [PMC free article] [PubMed]
  • Zeitlin S, Wilson RC, Efstratiadis A. Autonomous splicing and complementation of in vivo-assembled spliceosomes. J Cell Biol. 1989 Mar;108(3):765–777. [PMC free article] [PubMed]
  • Zeng C, He D, Berget SM, Brinkley BR. Nuclear-mitotic apparatus protein: a structural protein interface between the nucleoskeleton and RNA splicing. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1505–1509. [PMC free article] [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...