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. 1993 Dec 1;123(5):1055–1068. doi: 10.1083/jcb.123.5.1055

Nuclear organization of splicing snRNPs during differentiation of murine erythroleukemia cells in vitro

PMCID: PMC2119874  PMID: 8245117

Abstract

Murine erythroleukemia (MEL) cells are erythroid progenitors that can be induced to undergo terminal erythroid differentiation in culture. We have used MEL cells here as a model system to study the nuclear organization of splicing snRNPs during the physiological changes in gene expression which accompany differentiation. In uninduced MEL cells, snRNPs are widely distributed throughout the nucleoplasm and show an elevated concentration in coiled bodies. Within the first two days after induction of terminal erythroid differentiation, the pattern of gene expression changes, erythroid-specific transcription is activated and transcription of many other genes is repressed. During this early stage splicing snRNPs remain widely distributed through the nucleoplasm and continue to associate with coiled bodies. At later stages of differentiation (four to six days), when total transcription levels have greatly decreased, splicing snRNPs are redistributed. By six days postinduction snRNPs were concentrated in large clusters of interchromatin granules and no longer associated with coiled bodies. At the end-point of erythroid differentiation, just before enucleation, we observe a dramatic segregation of splicing snRNPs from the condensed chromatin. Analysis by EM shows that the snRNPs are packaged into a membrane-associated structure at the nuclear periphery which we term the "SCIM" domain (i.e., SnRNP Clusters Inside a Membrane).

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Selected References

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  1. Andrade L. E., Chan E. K., Raska I., Peebles C. L., Roos G., Tan E. M. Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin. J Exp Med. 1991 Jun 1;173(6):1407–1419. doi: 10.1084/jem.173.6.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Antoniou M., Grosveld F. beta-globin dominant control region interacts differently with distal and proximal promoter elements. Genes Dev. 1990 Jun;4(6):1007–1013. doi: 10.1101/gad.4.6.1007. [DOI] [PubMed] [Google Scholar]
  3. Bernhard W. A new staining procedure for electron microscopical cytology. J Ultrastruct Res. 1969 May;27(3):250–265. doi: 10.1016/s0022-5320(69)80016-x. [DOI] [PubMed] [Google Scholar]
  4. Beyer A. L., Osheim Y. N. Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. Genes Dev. 1988 Jun;2(6):754–765. doi: 10.1101/gad.2.6.754. [DOI] [PubMed] [Google Scholar]
  5. Billings P. B., Allen R. W., Jensen F. C., Hoch S. O. Anti-RNP monoclonal antibodies derived from a mouse strain with lupus-like autoimmunity. J Immunol. 1982 Mar;128(3):1176–1180. [PubMed] [Google Scholar]
  6. Carmo-Fonseca M., Pepperkok R., Carvalho M. T., Lamond A. I. Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 snRNPs in coiled bodies. J Cell Biol. 1992 Apr;117(1):1–14. doi: 10.1083/jcb.117.1.1. [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. Carmo-Fonseca M., Tollervey D., Pepperkok R., Barabino S. M., Merdes A., Brunner C., Zamore P. D., Green M. R., Hurt E., Lamond A. I. Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery. EMBO J. 1991 Jan;10(1):195–206. doi: 10.1002/j.1460-2075.1991.tb07936.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carter K. C., Bowman D., Carrington W., Fogarty K., McNeil J. A., Fay F. S., Lawrence J. B. A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. Science. 1993 Feb 26;259(5099):1330–1335. doi: 10.1126/science.8446902. [DOI] [PubMed] [Google Scholar]
  10. Collis P., Antoniou M., Grosveld F. Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression. EMBO J. 1990 Jan;9(1):233–240. doi: 10.1002/j.1460-2075.1990.tb08100.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deng J. S., Takasaki Y., Tan E. M. Nonhistone nuclear antigens reactive with autoantibodies. Immunofluorescence studies on distribution in synchronized cells. J Cell Biol. 1981 Dec;91(3 Pt 1):654–660. doi: 10.1083/jcb.91.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Djabali K., Portier M. M., Gros F., Blobel G., Georgatos S. D. Network antibodies identify nuclear lamin B as a physiological attachment site for peripherin intermediate filaments. Cell. 1991 Jan 11;64(1):109–121. doi: 10.1016/0092-8674(91)90213-i. [DOI] [PubMed] [Google Scholar]
  13. Fakan S., Leser G., Martin T. E. Ultrastructural distribution of nuclear ribonucleoproteins as visualized by immunocytochemistry on thin sections. J Cell Biol. 1984 Jan;98(1):358–363. doi: 10.1083/jcb.98.1.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Friend C., Scher W., Holland J. G., Sato T. Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide. Proc Natl Acad Sci U S A. 1971 Feb;68(2):378–382. doi: 10.1073/pnas.68.2.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fu X. D., Maniatis T. Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature. 1990 Feb 1;343(6257):437–441. doi: 10.1038/343437a0. [DOI] [PubMed] [Google Scholar]
  16. Habets W. J., Hoet M. H., De Jong B. A., Van der Kemp A., Van Venrooij W. J. Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies. J Immunol. 1989 Oct 15;143(8):2560–2566. [PubMed] [Google Scholar]
  17. Huang S., Spector D. L. Nascent pre-mRNA transcripts are associated with nuclear regions enriched in splicing factors. Genes Dev. 1991 Dec;5(12A):2288–2302. doi: 10.1101/gad.5.12a.2288. [DOI] [PubMed] [Google Scholar]
  18. Huang S., Spector D. L. 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. doi: 10.1073/pnas.89.1.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Krol A., Carbon P., Ebel J. P., Appel B. Xenopus tropicalis U6 snRNA genes transcribed by Pol III contain the upstream promoter elements used by Pol II dependent U snRNA genes. Nucleic Acids Res. 1987 Mar 25;15(6):2463–2478. doi: 10.1093/nar/15.6.2463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lamond A. I., Carmo-Fonseca M. The coiled body. Trends Cell Biol. 1993 Jun;3(6):198–204. doi: 10.1016/0962-8924(93)90214-l. [DOI] [PubMed] [Google Scholar]
  21. Lawrence J. B., Singer R. H., Marselle L. M. Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell. 1989 May 5;57(3):493–502. doi: 10.1016/0092-8674(89)90924-0. [DOI] [PubMed] [Google Scholar]
  22. Lerner E. A., Lerner M. R., Janeway C. A., Jr, Steitz J. A. Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease. Proc Natl Acad Sci U S A. 1981 May;78(5):2737–2741. doi: 10.1073/pnas.78.5.2737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lerner M. R., Steitz J. A. Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5495–5499. doi: 10.1073/pnas.76.11.5495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leser G. P., Fakan S., Martin T. E. Ultrastructural distribution of ribonucleoprotein complexes during mitosis. snRNP antigens are contained in mitotic granule clusters. Eur J Cell Biol. 1989 Dec;50(2):376–389. [PubMed] [Google Scholar]
  25. Lührmann R., Kastner B., Bach M. Structure of spliceosomal snRNPs and their role in pre-mRNA splicing. Biochim Biophys Acta. 1990 Nov 30;1087(3):265–292. doi: 10.1016/0167-4781(90)90001-i. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Nyman U., Hallman H., Hadlaczky G., Pettersson I., Sharp G., Ringertz N. R. Intranuclear localization of snRNP antigens. J Cell Biol. 1986 Jan;102(1):137–144. doi: 10.1083/jcb.102.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Orkin S. H., Swerdlow P. S. Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: direct evidence for increased transcription during erythroid differentiation. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2475–2479. doi: 10.1073/pnas.74.6.2475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Patel V. P., Lodish H. F. A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes. J Cell Biol. 1987 Dec;105(6 Pt 2):3105–3118. doi: 10.1083/jcb.105.6.3105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Patel V. P., Lodish H. F. Loss of adhesion of murine erythroleukemia cells to fibronectin during erythroid differentiation. Science. 1984 Jun 1;224(4652):996–998. doi: 10.1126/science.6585955. [DOI] [PubMed] [Google Scholar]
  31. Pettersson I., Hinterberger M., Mimori T., Gottlieb E., Steitz J. A. The structure of mammalian small nuclear ribonucleoproteins. Identification of multiple protein components reactive with anti-(U1)ribonucleoprotein and anti-Sm autoantibodies. J Biol Chem. 1984 May 10;259(9):5907–5914. [PubMed] [Google Scholar]
  32. Puvion E., Viron A., Assens C., Leduc E. H., Jeanteur P. Immunocytochemical identification of nuclear structures containing snRNPs in isolated rat liver cells. J Ultrastruct Res. 1984 May;87(2):180–189. doi: 10.1016/s0022-5320(84)80077-5. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Reuter R., Appel B., Bringmann P., Rinke J., Lührmann R. 5'-Terminal caps of snRNAs are reactive with antibodies specific for 2,2,7-trimethylguanosine in whole cells and nuclear matrices. Double-label immunofluorescent studies with anti-m3G antibodies and with anti-RNP and anti-Sm autoantibodies. Exp Cell Res. 1984 Oct;154(2):548–560. doi: 10.1016/0014-4827(84)90179-4. [DOI] [PubMed] [Google Scholar]
  35. Sherton C. C., Kabat D. Changes in RNA and protein metabolism preceding onset of hemoglobin synthesis in cultured Friend leukemia cells. Dev Biol. 1976 Jan;48(1):118–131. doi: 10.1016/0012-1606(76)90051-8. [DOI] [PubMed] [Google Scholar]
  36. Singer D., Cooper M., Maniatis G. M., Marks P. A., Rifkind R. A. Erythropoietic differentiation in colonies of cells transformed by Friend virus. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2668–2670. doi: 10.1073/pnas.71.7.2668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Spector D. L. Colocalization of U1 and U2 small nuclear RNPs by immunocytochemistry. Biol Cell. 1984;51(1):109–112. doi: 10.1111/j.1768-322x.1984.tb00289.x. [DOI] [PubMed] [Google Scholar]
  38. Spector D. L., Fu X. D., Maniatis T. Associations between distinct pre-mRNA splicing components and the cell nucleus. EMBO J. 1991 Nov;10(11):3467–3481. doi: 10.1002/j.1460-2075.1991.tb04911.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Spector D. L. 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. doi: 10.1073/pnas.87.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takagaki Y., Manley J. L., MacDonald C. C., Wilusz J., Shenk T. A multisubunit factor, CstF, is required for polyadenylation of mammalian pre-mRNAs. Genes Dev. 1990 Dec;4(12A):2112–2120. doi: 10.1101/gad.4.12a.2112. [DOI] [PubMed] [Google Scholar]
  41. Turner B. M., 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. doi: 10.1242/jcs.87.2.269. [DOI] [PubMed] [Google Scholar]
  42. Verheijen R., Kuijpers H., Vooijs P., Van Venrooij W., Ramaekers F. Distribution of the 70K U1 RNA-associated protein during interphase and mitosis. Correlation with other U RNP particles and proteins of the nuclear matrix. J Cell Sci. 1986 Dec;86:173–190. doi: 10.1242/jcs.86.1.173. [DOI] [PubMed] [Google Scholar]
  43. Visa N., Puvion-Dutilleul F., Bachellerie J. P., Puvion E. Intranuclear distribution of U1 and U2 snRNAs visualized by high resolution in situ hybridization: revelation of a novel compartment containing U1 but not U2 snRNA in HeLa cells. Eur J Cell Biol. 1993 Apr;60(2):308–321. [PubMed] [Google Scholar]
  44. Volloch V., Housman D. Terminal differentiation of murine erythroleukemia cells: physical stabilization of end-stage cells. J Cell Biol. 1982 May;93(2):390–394. doi: 10.1083/jcb.93.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Xing Y., Johnson C. V., Dobner P. R., Lawrence J. B. Higher level organization of individual gene transcription and RNA splicing. Science. 1993 Feb 26;259(5099):1326–1330. doi: 10.1126/science.8446901. [DOI] [PubMed] [Google Scholar]
  46. Zamore P. D., Green M. R. Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution. EMBO J. 1991 Jan;10(1):207–214. doi: 10.1002/j.1460-2075.1991.tb07937.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhang M., Zamore P. D., Carmo-Fonseca M., Lamond A. I., Green M. R. Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8769–8773. doi: 10.1073/pnas.89.18.8769. [DOI] [PMC free article] [PubMed] [Google Scholar]

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