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. 1999 Apr 15;18(8):2241–2253. doi: 10.1093/emboj/18.8.2241

Regional specialization in human nuclei: visualization of discrete sites of transcription by RNA polymerase III.

A Pombo 1, D A Jackson 1, M Hollinshead 1, Z Wang 1, R G Roeder 1, P R Cook 1
PMCID: PMC1171307  PMID: 10205177

Abstract

Mammalian nuclei contain three different RNA polymerases defined by their characteristic locations and drug sensitivities; polymerase I is found in nucleoli, and polymerases II and III in the nucleoplasm. As nascent transcripts made by polymerases I and II are concentrated in discrete sites, the locations of those made by polymerase III were investigated. HeLa cells were lysed with saponin in an improved 'physiological' buffer that preserves transcriptional activity and nuclear ultrastructure; then, engaged polymerases were allowed to extend nascent transcripts in Br-UTP, before the resulting Br-RNA was immunolabelled indirectly with fluorochromes or gold particles. Biochemical analysis showed that approximately 10 000 transcripts were being made by polymerase III at the moment of lysis, while confocal and electron microscopy showed that these transcripts were concentrated in only approximately 2000 sites (diameter approximately 40 nm). Therefore, each site contains approximately five active polymerases. These sites contain specific subunits of polymerase III, but not the hyperphosphorylated form of the largest subunit of polymerase II. The results indicate that the active forms of all three nuclear polymerases are concentrated in their own dedicated transcription sites or 'factories', suggesting that different regions of the nucleus specialize in the transcription of different types of gene.

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Selected References

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  1. Bartholomew B., Durkovich D., Kassavetis G. A., Geiduschek E. P. Orientation and topography of RNA polymerase III in transcription complexes. Mol Cell Biol. 1993 Feb;13(2):942–952. doi: 10.1128/mcb.13.2.942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Braun B. R., Riggs D. L., Kassavetis G. A., Geiduschek E. P. Multiple states of protein-DNA interaction in the assembly of transcription complexes on Saccharomyces cerevisiae 5S ribosomal RNA genes. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2530–2534. doi: 10.1073/pnas.86.8.2530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bregman D. B., Du L., van der Zee S., Warren S. L. Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains. J Cell Biol. 1995 Apr;129(2):287–298. doi: 10.1083/jcb.129.2.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dahmus M. E. Reversible phosphorylation of the C-terminal domain of RNA polymerase II. J Biol Chem. 1996 Aug 9;271(32):19009–19012. doi: 10.1074/jbc.271.32.19009. [DOI] [PubMed] [Google Scholar]
  5. Fakan S. Perichromatin fibrils are in situ forms of nascent transcripts. Trends Cell Biol. 1994 Mar;4(3):86–90. doi: 10.1016/0962-8924(94)90180-5. [DOI] [PubMed] [Google Scholar]
  6. Fakan S., Puvion E., Sphor G. Localization and characterization of newly synthesized nuclear RNA in isolate rat hepatocytes. Exp Cell Res. 1976 Apr;99(1):155–164. doi: 10.1016/0014-4827(76)90690-x. [DOI] [PubMed] [Google Scholar]
  7. Fakan S., Puvion E. The ultrastructural visualization of nucleolar and extranucleolar RNA synthesis and distribution. Int Rev Cytol. 1980;65:255–299. doi: 10.1016/s0074-7696(08)61962-2. [DOI] [PubMed] [Google Scholar]
  8. Fay F. S., Taneja K. L., Shenoy S., Lifshitz L., Singer R. H. Quantitative digital analysis of diffuse and concentrated nuclear distributions of nascent transcripts, SC35 and poly(A). Exp Cell Res. 1997 Feb 25;231(1):27–37. doi: 10.1006/excr.1996.3460. [DOI] [PubMed] [Google Scholar]
  9. Geiduschek E. P., Tocchini-Valentini G. P. Transcription by RNA polymerase III. Annu Rev Biochem. 1988;57:873–914. doi: 10.1146/annurev.bi.57.070188.004301. [DOI] [PubMed] [Google Scholar]
  10. Grande M. A., van der Kraan I., de Jong L., van Driel R. Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II. J Cell Sci. 1997 Aug;110(Pt 15):1781–1791. doi: 10.1242/jcs.110.15.1781. [DOI] [PubMed] [Google Scholar]
  11. Griffiths G., McDowall A., Back R., Dubochet J. On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res. 1984 Oct;89(1):65–78. doi: 10.1016/s0022-5320(84)80024-6. [DOI] [PubMed] [Google Scholar]
  12. Hatlen L., Attardi G. Proportion of HeLa cell genome complementary to transfer RNA and 5 s RNA. J Mol Biol. 1971 Mar 28;56(3):535–553. doi: 10.1016/0022-2836(71)90400-1. [DOI] [PubMed] [Google Scholar]
  13. Hozák P., Cook P. R., Schöfer C., Mosgöller W., Wachtler F. Site of transcription of ribosomal RNA and intranucleolar structure in HeLa cells. J Cell Sci. 1994 Feb;107(Pt 2):639–648. doi: 10.1242/jcs.107.2.639. [DOI] [PubMed] [Google Scholar]
  14. Iborra F. J., Cook P. R. The size of sites containing SR proteins in human nuclei. Problems associated with characterizing small structures by immunogold labeling. J Histochem Cytochem. 1998 Sep;46(9):985–992. doi: 10.1177/002215549804600901. [DOI] [PubMed] [Google Scholar]
  15. Iborra F. J., Jackson D. A., Cook P. R. The path of transcripts from extra-nucleolar synthetic sites to nuclear pores: transcripts in transit are concentrated in discrete structures containing SR proteins. J Cell Sci. 1998 Aug;111(Pt 15):2269–2282. doi: 10.1242/jcs.111.15.2269. [DOI] [PubMed] [Google Scholar]
  16. Iborra F. J., Pombo A., Jackson D. A., Cook P. R. Active RNA polymerases are localized within discrete transcription "factories' in human nuclei. J Cell Sci. 1996 Jun;109(Pt 6):1427–1436. doi: 10.1242/jcs.109.6.1427. [DOI] [PubMed] [Google Scholar]
  17. Jackson D. A., Cook P. R. Transcription occurs at a nucleoskeleton. EMBO J. 1985 Apr;4(4):919–925. doi: 10.1002/j.1460-2075.1985.tb03719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jackson D. A., Hassan A. B., Errington R. J., Cook P. R. Visualization of focal sites of transcription within human nuclei. EMBO J. 1993 Mar;12(3):1059–1065. doi: 10.1002/j.1460-2075.1993.tb05747.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jackson D. A., Iborra F. J., Manders E. M., Cook P. R. Numbers and organization of RNA polymerases, nascent transcripts, and transcription units in HeLa nuclei. Mol Biol Cell. 1998 Jun;9(6):1523–1536. doi: 10.1091/mbc.9.6.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jackson D. A., Yuan J., Cook P. R. A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin. J Cell Sci. 1988 Jul;90(Pt 3):365–378. doi: 10.1242/jcs.90.3.365. [DOI] [PubMed] [Google Scholar]
  21. Kim E., Du L., Bregman D. B., Warren S. L. Splicing factors associate with hyperphosphorylated RNA polymerase II in the absence of pre-mRNA. J Cell Biol. 1997 Jan 13;136(1):19–28. doi: 10.1083/jcb.136.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kovelman R., Roeder R. G. Sarkosyl defines three intermediate steps in transcription initiation by RNA polymerase III: application to stimulation of transcription by E1A. Genes Dev. 1990 Apr;4(4):646–658. doi: 10.1101/gad.4.4.646. [DOI] [PubMed] [Google Scholar]
  23. Köck J., Cornelissen A. W. Characterization of the RNA polymerases of Crithidia fasciculata. Mol Microbiol. 1991 Apr;5(4):835–842. doi: 10.1111/j.1365-2958.1991.tb00756.x. [DOI] [PubMed] [Google Scholar]
  24. Lea P., Gross D. K. Effective diameters of protein A-gold and goat anti-rabbit-gold conjugates visualized by field emission scanning electron microscopy. J Histochem Cytochem. 1992 Jun;40(6):751–758. doi: 10.1177/40.6.1588022. [DOI] [PubMed] [Google Scholar]
  25. Little R. D., Braaten D. C. Genomic organization of human 5 S rDNA and sequence of one tandem repeat. Genomics. 1989 Apr;4(3):376–383. doi: 10.1016/0888-7543(89)90345-5. [DOI] [PubMed] [Google Scholar]
  26. Maraia R. J., Kenan D. J., Keene J. D. Eukaryotic transcription termination factor La mediates transcript release and facilitates reinitiation by RNA polymerase III. Mol Cell Biol. 1994 Mar;14(3):2147–2158. doi: 10.1128/mcb.14.3.2147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marzluff W. F., Jr, Murphy E. C., Jr, Huang R. C. Transcription of the genes for 5S ribosomal RNA and transfer RNA in isolated mouse myeloma cell nuclei. Biochemistry. 1974 Aug 27;13(18):3689–3696. doi: 10.1021/bi00715a011. [DOI] [PubMed] [Google Scholar]
  28. Matera A. G., Frey M. R., Margelot K., Wolin S. L. A perinucleolar compartment contains several RNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I. J Cell Biol. 1995 Jun;129(5):1181–1193. doi: 10.1083/jcb.129.5.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matera A. G., Ward D. C. Nucleoplasmic organization of small nuclear ribonucleoproteins in cultured human cells. J Cell Biol. 1993 May;121(4):715–727. doi: 10.1083/jcb.121.4.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pombo A., Cook P. R. The localization of sites containing nascent RNA and splicing factors. Exp Cell Res. 1996 Dec 15;229(2):201–203. doi: 10.1006/excr.1996.0360. [DOI] [PubMed] [Google Scholar]
  31. Pombo A., Cuello P., Schul W., Yoon J. B., Roeder R. G., Cook P. R., Murphy S. Regional and temporal specialization in the nucleus: a transcriptionally-active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle. EMBO J. 1998 Mar 16;17(6):1768–1778. doi: 10.1093/emboj/17.6.1768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pombo A., Ferreira J., Bridge E., Carmo-Fonseca M. Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells. EMBO J. 1994 Nov 1;13(21):5075–5085. doi: 10.1002/j.1460-2075.1994.tb06837.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pombo A., Hollinshead M., Cook P. R. Bridging the resolution gap: Imaging the same transcription factories in cryosections by light and electron microscopy. J Histochem Cytochem. 1999 Apr;47(4):471–480. doi: 10.1177/002215549904700405. [DOI] [PubMed] [Google Scholar]
  34. Shaw P. J., Jordan E. G. The nucleolus. Annu Rev Cell Dev Biol. 1995;11:93–121. doi: 10.1146/annurev.cb.11.110195.000521. [DOI] [PubMed] [Google Scholar]
  35. Steinberg T. H., Burgess R. R. Tagetitoxin inhibition of RNA polymerase III transcription results from enhanced pausing at discrete sites and is template-dependent. J Biol Chem. 1992 Oct 5;267(28):20204–20211. [PubMed] [Google Scholar]
  36. Steinberg T. H., Mathews D. E., Durbin R. D., Burgess R. R. Tagetitoxin: a new inhibitor of eukaryotic transcription by RNA polymerase III. J Biol Chem. 1990 Jan 5;265(1):499–505. [PubMed] [Google Scholar]
  37. Sørensen P. D., Frederiksen S. Characterization of human 5S rRNA genes. Nucleic Acids Res. 1991 Aug 11;19(15):4147–4151. doi: 10.1093/nar/19.15.4147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  39. Tooze J., Hollinshead M., Hensel G., Kern H. F., Hoflack B. Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells. Eur J Cell Biol. 1991 Dec;56(2):187–200. [PubMed] [Google Scholar]
  40. Tooze J., Hollinshead M. In AtT20 and HeLa cells brefeldin A induces the fusion of tubular endosomes and changes their distribution and some of their endocytic properties. J Cell Biol. 1992 Aug;118(4):813–830. doi: 10.1083/jcb.118.4.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Udvardy A., Seifart K. H. Transcription of specific genes in isolated nuclei from HeLa cells in vitro. Eur J Biochem. 1976 Feb 16;62(2):353–363. doi: 10.1111/j.1432-1033.1976.tb10167.x. [DOI] [PubMed] [Google Scholar]
  42. Wang Z., Roeder R. G. Three human RNA polymerase III-specific subunits form a subcomplex with a selective function in specific transcription initiation. Genes Dev. 1997 May 15;11(10):1315–1326. doi: 10.1101/gad.11.10.1315. [DOI] [PubMed] [Google Scholar]
  43. Wansink D. G., 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. doi: 10.1083/jcb.122.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weil P. A., Blatti S. P. HeLa cell deoxyribonucleic acid dependent RNA polymerases: function and properties of the class III enzymes. Biochemistry. 1976 Apr 6;15(7):1500–1509. doi: 10.1021/bi00652a022. [DOI] [PubMed] [Google Scholar]
  45. Weinmann R., Raskas H. J., Roeder R. G. The transcriptional role of host DNA-dependent RNA polymerases in adenovirus-infected KB cells. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):495–499. doi: 10.1101/sqb.1974.039.01.061. [DOI] [PubMed] [Google Scholar]
  46. Zeng C., Kim E., Warren S. L., Berget S. M. Dynamic relocation of transcription and splicing factors dependent upon transcriptional activity. EMBO J. 1997 Mar 17;16(6):1401–1412. doi: 10.1093/emboj/16.6.1401. [DOI] [PMC free article] [PubMed] [Google Scholar]

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