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. 1994 Dec;5(12):1289–1299. doi: 10.1091/mbc.5.12.1289

Organization of small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in situ hybridization and immunocytochemistry.

A G Matera 1, K T Tycowski 1, J A Steitz 1, D C Ward 1
PMCID: PMC301158  PMID: 7535131

Abstract

The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baserga S. J., Yang X. D., Steitz J. A. An intact Box C sequence in the U3 snRNA is required for binding of fibrillarin, the protein common to the major family of nucleolar snRNPs. EMBO J. 1991 Sep;10(9):2645–2651. doi: 10.1002/j.1460-2075.1991.tb07807.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauer D. W., Murphy C., Wu Z., Wu C. H., Gall J. G. In vitro assembly of coiled bodies in Xenopus egg extract. Mol Biol Cell. 1994 Jun;5(6):633–644. doi: 10.1091/mbc.5.6.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bell P., Dabauvalle M. C., Scheer U. In vitro assembly of prenucleolar bodies in Xenopus egg extract. J Cell Biol. 1992 Sep;118(6):1297–1304. doi: 10.1083/jcb.118.6.1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beltrame M., Tollervey D. Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA. EMBO J. 1992 Apr;11(4):1531–1542. doi: 10.1002/j.1460-2075.1992.tb05198.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benavente R., Rose K. M., Reimer G., Hügle-Dörr B., Scheer U. Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells. J Cell Biol. 1987 Oct;105(4):1483–1491. doi: 10.1083/jcb.105.4.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carmo-Fonseca M., Ferreira J., Lamond A. I. Assembly of snRNP-containing coiled bodies is regulated in interphase and mitosis--evidence that the coiled body is a kinetic nuclear structure. J Cell Biol. 1993 Feb;120(4):841–852. doi: 10.1083/jcb.120.4.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carmo-Fonseca M., Pepperkok R., Sproat B. S., Ansorge W., Swanson M. S., Lamond A. I. In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells. EMBO J. 1991 Jul;10(7):1863–1873. doi: 10.1002/j.1460-2075.1991.tb07712.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chan E. K., Imai H., Hamel J. C., Tan E. M. Human autoantibody to RNA polymerase I transcription factor hUBF. Molecular identity of nucleolus organizer region autoantigen NOR-90 and ribosomal RNA transcription upstream binding factor. J Exp Med. 1991 Nov 1;174(5):1239–1244. doi: 10.1084/jem.174.5.1239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Filipowicz W., Kiss T. Structure and function of nucleolar snRNPs. Mol Biol Rep. 1993 Aug;18(2):149–156. doi: 10.1007/BF00986770. [DOI] [PubMed] [Google Scholar]
  10. Fischer D., Weisenberger D., Scheer U. Assigning functions to nucleolar structures. Chromosoma. 1991 Dec;101(3):133–140. doi: 10.1007/BF00355363. [DOI] [PubMed] [Google Scholar]
  11. Fournier M. J., Maxwell E. S. The nucleolar snRNAs: catching up with the spliceosomal snRNAs. Trends Biochem Sci. 1993 Apr;18(4):131–135. doi: 10.1016/0968-0004(93)90020-n. [DOI] [PubMed] [Google Scholar]
  12. Haaf T., Hayman D. L., Schmid M. Quantitative determination of rDNA transcription units in vertebrate cells. Exp Cell Res. 1991 Mar;193(1):78–86. doi: 10.1016/0014-4827(91)90540-b. [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. Huang G. M., Jarmolowski A., Struck J. C., Fournier M. J. Accumulation of U14 small nuclear RNA in Saccharomyces cerevisiae requires box C, box D, and a 5', 3' terminal stem. Mol Cell Biol. 1992 Oct;12(10):4456–4463. doi: 10.1128/mcb.12.10.4456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hughes J. M., Ares M., Jr Depletion of U3 small nucleolar RNA inhibits cleavage in the 5' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA. EMBO J. 1991 Dec;10(13):4231–4239. doi: 10.1002/j.1460-2075.1991.tb05001.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jiménez-García L. F., Segura-Valdez M. L., Ochs R. L., Rothblum L. I., Hannan R., Spector D. L. Nucleologenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis. Mol Biol Cell. 1994 Sep;5(9):955–966. doi: 10.1091/mbc.5.9.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Karpen G. H., Schaefer J. E., Laird C. D. A Drosophila rRNA gene located in euchromatin is active in transcription and nucleolus formation. Genes Dev. 1988 Dec;2(12B):1745–1763. doi: 10.1101/gad.2.12b.1745. [DOI] [PubMed] [Google Scholar]
  18. Kass S., Tyc K., Steitz J. A., Sollner-Webb B. The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing. Cell. 1990 Mar 23;60(6):897–908. doi: 10.1016/0092-8674(90)90338-f. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Matera A. G., Ward D. C. Oligonucleotide probes for the analysis of specific repetitive DNA sequences by fluorescence in situ hybridization. Hum Mol Genet. 1992 Oct;1(7):535–539. doi: 10.1093/hmg/1.7.535. [DOI] [PubMed] [Google Scholar]
  21. Mougey E. B., O'Reilly M., Osheim Y., Miller O. L., Jr, Beyer A., Sollner-Webb B. The terminal balls characteristic of eukaryotic rRNA transcription units in chromatin spreads are rRNA processing complexes. Genes Dev. 1993 Aug;7(8):1609–1619. doi: 10.1101/gad.7.8.1609. [DOI] [PubMed] [Google Scholar]
  22. Mougey E. B., Pape L. K., Sollner-Webb B. A U3 small nuclear ribonucleoprotein-requiring processing event in the 5' external transcribed spacer of Xenopus precursor rRNA. Mol Cell Biol. 1993 Oct;13(10):5990–5998. doi: 10.1128/mcb.13.10.5990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Peculis B. A., Steitz J. A. Disruption of U8 nucleolar snRNA inhibits 5.8S and 28S rRNA processing in the Xenopus oocyte. Cell. 1993 Jun 18;73(6):1233–1245. doi: 10.1016/0092-8674(93)90651-6. [DOI] [PubMed] [Google Scholar]
  24. Peculis B. A., Steitz J. A. Sequence and structural elements critical for U8 snRNP function in Xenopus oocytes are evolutionarily conserved. Genes Dev. 1994 Sep 15;8(18):2241–2255. doi: 10.1101/gad.8.18.2241. [DOI] [PubMed] [Google Scholar]
  25. Puvion-Dutilleul F., Mazan S., Nicoloso M., Christensen M. E., Bachellerie J. P. Localization of U3 RNA molecules in nucleoli of HeLa and mouse 3T3 cells by high resolution in situ hybridization. Eur J Cell Biol. 1991 Dec;56(2):178–186. [PubMed] [Google Scholar]
  26. Raska I., Andrade L. E., Ochs R. L., Chan E. K., Chang C. M., Roos G., Tan E. M. Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies. Exp Cell Res. 1991 Jul;195(1):27–37. doi: 10.1016/0014-4827(91)90496-h. [DOI] [PubMed] [Google Scholar]
  27. Raska I., Ochs R. L., Andrade L. E., Chan E. K., Burlingame R., Peebles C., Gruol D., Tan E. M. Association between the nucleolus and the coiled body. J Struct Biol. 1990 Jul-Sep;104(1-3):120–127. doi: 10.1016/1047-8477(90)90066-l. [DOI] [PubMed] [Google Scholar]
  28. Reeder R. H. rRNA synthesis in the nucleolus. Trends Genet. 1990 Dec;6(12):390–395. doi: 10.1016/0168-9525(90)90298-k. [DOI] [PubMed] [Google Scholar]
  29. Reimer G., Raska I., Tan E. M., Scheer U. Human autoantibodies: probes for nucleolus structure and function. Virchows Arch B Cell Pathol Incl Mol Pathol. 1987;54(3):131–143. doi: 10.1007/BF02899205. [DOI] [PubMed] [Google Scholar]
  30. Reimer G., Rose K. M., Scheer U., Tan E. M. Autoantibody to RNA polymerase I in scleroderma sera. J Clin Invest. 1987 Jan;79(1):65–72. doi: 10.1172/JCI112809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rendón M. C., Rodrigo R. M., Goenechea L. G., García-Herdugo G., Valdivia M. M., Moreno F. J. Characterization and immunolocalization of a nucleolar antigen with anti-NOR serum in HeLa cells. Exp Cell Res. 1992 Jun;200(2):393–403. doi: 10.1016/0014-4827(92)90187-d. [DOI] [PubMed] [Google Scholar]
  32. Rose K. M., Szopa J., Han F. S., Cheng Y. C., Richter A., Scheer U. Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription. Chromosoma. 1988;96(6):411–416. doi: 10.1007/BF00303034. [DOI] [PubMed] [Google Scholar]
  33. Savino R., Gerbi S. A. In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing. EMBO J. 1990 Jul;9(7):2299–2308. doi: 10.1002/j.1460-2075.1990.tb07401.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Scheer U., Rose K. M. Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1431–1435. doi: 10.1073/pnas.81.5.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sollner-Webb B. Novel intron-encoded small nucleolar RNAs. Cell. 1993 Nov 5;75(3):403–405. doi: 10.1016/0092-8674(93)90374-y. [DOI] [PubMed] [Google Scholar]
  36. Stevens A. R., Prescott D. M. Reformation of nucleolus-like bodies in the absence of postmitotic RNA synthesis. J Cell Biol. 1971 Mar;48(3):443–454. doi: 10.1083/jcb.48.3.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stroke I. L., Weiner A. M. The 5' end of U3 snRNA can be crosslinked in vivo to the external transcribed spacer of rat ribosomal RNA precursors. J Mol Biol. 1989 Dec 5;210(3):497–512. doi: 10.1016/0022-2836(89)90126-5. [DOI] [PubMed] [Google Scholar]
  38. Tyc K., Steitz J. A. U3, U8 and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus. EMBO J. 1989 Oct;8(10):3113–3119. doi: 10.1002/j.1460-2075.1989.tb08463.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Voit R., Schnapp A., Kuhn A., Rosenbauer H., Hirschmann P., Stunnenberg H. G., Grummt I. The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation. EMBO J. 1992 Jun;11(6):2211–2218. doi: 10.1002/j.1460-2075.1992.tb05280.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Warner J. R. The nucleolus and ribosome formation. Curr Opin Cell Biol. 1990 Jun;2(3):521–527. doi: 10.1016/0955-0674(90)90137-4. [DOI] [PubMed] [Google Scholar]
  41. Zatsepina O. V., Voit R., Grummt I., Spring H., Semenov M. V., Trendelenburg M. F. The RNA polymerase I-specific transcription initiation factor UBF is associated with transcriptionally active and inactive ribosomal genes. Chromosoma. 1993 Nov;102(9):599–611. doi: 10.1007/BF00352307. [DOI] [PubMed] [Google Scholar]

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