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. 1991 Jan;10(1):195–206. doi: 10.1002/j.1460-2075.1991.tb07936.x

Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery.

M Carmo-Fonseca 1, D Tollervey 1, R Pepperkok 1, S M Barabino 1, A Merdes 1, C Brunner 1, P D Zamore 1, M R Green 1, E Hurt 1, A I Lamond 1
PMCID: PMC452630  PMID: 1824936

Abstract

The organization of the major snRNP particles in mammalian cell nuclei has been analysed by in situ labelling using snRNA-specific antisense probes made of 2'-OMe RNA. U3 snRNA is exclusively detected in the nucleolus while all the spliceosomal snRNAs are found in the nucleoplasm outside of nucleoli. Surprisingly, U2, U4, U5 and U6 snRNAs are predominantly observed in discrete nucleoplasmic foci. U1 snRNA is also present in foci but in addition is detected widely distributed throughout the nucleoplasm. An anti-peptide antibody specific for the non-snRNP splicing factor U2AF reveals it to have a similar distribution to U1 snRNA. Co-localization studies using confocal fluorescence microscopy prove that U2AF is present in the snRNA-containing foci. Antibody staining also shows the foci to contain snRNP-specific proteins and m3G-cap structures. The presence of major components of the nuclear splicing apparatus in foci suggests that these structures may play a role in pre-mRNA processing.

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Selected References

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  1. Barabino S. M., Blencowe B. J., Ryder U., Sproat B. S., Lamond A. I. Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly. Cell. 1990 Oct 19;63(2):293–302. doi: 10.1016/0092-8674(90)90162-8. [DOI] [PubMed] [Google Scholar]
  2. Barabino S. M., Sproat B. S., Ryder U., Blencowe B. J., Lamond A. I. Mapping U2 snRNP--pre-mRNA interactions using biotinylated oligonucleotides made of 2'-OMe RNA. EMBO J. 1989 Dec 20;8(13):4171–4178. doi: 10.1002/j.1460-2075.1989.tb08602.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bennett F. C., Yeoman L. C. Co-localization of non-histone protein BA with U-snRNPs to the same regions of the cell nucleus. Exp Cell Res. 1985 Apr;157(2):379–386. doi: 10.1016/0014-4827(85)90123-5. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Black D. L., Pinto A. L. U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6. Mol Cell Biol. 1989 Aug;9(8):3350–3359. doi: 10.1128/mcb.9.8.3350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blencowe B. J., Sproat B. S., Ryder U., Barabino S., Lamond A. I. Antisense probing of the human U4/U6 snRNP with biotinylated 2'-OMe RNA oligonucleotides. Cell. 1989 Nov 3;59(3):531–539. doi: 10.1016/0092-8674(89)90036-6. [DOI] [PubMed] [Google Scholar]
  7. Bringmann P., Appel B., Rinke J., Reuter R., Theissen H., Lührmann R. Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing. EMBO J. 1984 Jun;3(6):1357–1363. doi: 10.1002/j.1460-2075.1984.tb01977.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng S. C., Abelson J. Spliceosome assembly in yeast. Genes Dev. 1987 Nov;1(9):1014–1027. doi: 10.1101/gad.1.9.1014. [DOI] [PubMed] [Google Scholar]
  9. Choi Y. D., Dreyfuss G. Monoclonal antibody characterization of the C proteins of heterogeneous nuclear ribonucleoprotein complexes in vertebrate cells. J Cell Biol. 1984 Dec;99(6):1997–1204. doi: 10.1083/jcb.99.6.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fakan S., Leser G., Martin T. E. Immunoelectron microscope visualization of nuclear ribonucleoprotein antigens within spread transcription complexes. J Cell Biol. 1986 Oct;103(4):1153–1157. doi: 10.1083/jcb.103.4.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fey E. G., 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. doi: 10.1083/jcb.102.5.1654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fisher D. E., Conner G. E., Reeves W. H., Blobel G., Kunkel H. G. Synthesis and assembly of human small nuclear ribonucleoproteins generated by cell-free translation. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6356–6360. doi: 10.1073/pnas.80.20.6356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Gall J. G., Callan H. G. The sphere organelle contains small nuclear ribonucleoproteins. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6635–6639. doi: 10.1073/pnas.86.17.6635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gerke V., Steitz J. A. A protein associated with small nuclear ribonucleoprotein particles recognizes the 3' splice site of premessenger RNA. Cell. 1986 Dec 26;47(6):973–984. doi: 10.1016/0092-8674(86)90812-3. [DOI] [PubMed] [Google Scholar]
  17. Guthrie C., Patterson B. Spliceosomal snRNAs. Annu Rev Genet. 1988;22:387–419. doi: 10.1146/annurev.ge.22.120188.002131. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Hashimoto C., Steitz J. A. U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle. Nucleic Acids Res. 1984 Apr 11;12(7):3283–3293. doi: 10.1093/nar/12.7.3283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Iribarren A. M., Sproat B. S., Neuner P., Sulston I., Ryder U., Lamond A. I. 2'-O-alkyl oligoribonucleotides as antisense probes. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7747–7751. doi: 10.1073/pnas.87.19.7747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  23. Krainer A. R., Conway G. C., Kozak D. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 1990 Jul;4(7):1158–1171. doi: 10.1101/gad.4.7.1158. [DOI] [PubMed] [Google Scholar]
  24. Krämer A. Presplicing complex formation requires two proteins and U2 snRNP. Genes Dev. 1988 Sep;2(9):1155–1167. doi: 10.1101/gad.2.9.1155. [DOI] [PubMed] [Google Scholar]
  25. Lamond A. I., Sproat B., Ryder U., Hamm J. Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2'-OMe RNA. Cell. 1989 Jul 28;58(2):383–390. doi: 10.1016/0092-8674(89)90852-0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Luhrmann R., Appel B., Bringmann P., Rinke J., Reuter R., Rothe S., Bald R. Isolation and characterization of rabbit anti-m3 2,2,7G antibodies. Nucleic Acids Res. 1982 Nov 25;10(22):7103–7113. doi: 10.1093/nar/10.22.7103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Maniatis T., Reed R. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature. 1987 Feb 19;325(6106):673–678. doi: 10.1038/325673a0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Parker K. A., Steitz J. A. Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA. Mol Cell Biol. 1987 Aug;7(8):2899–2913. doi: 10.1128/mcb.7.8.2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Reuter R., Lehner C. F., Nigg E. A., Lührmann R. A monoclonal antibody specific for snRNPs U1 and U2. FEBS Lett. 1986 May 26;201(1):25–30. doi: 10.1016/0014-5793(86)80564-6. [DOI] [PubMed] [Google Scholar]
  35. Reuter R., Lührmann R. Immunization of mice with purified U1 small nuclear ribonucleoprotein (RNP) induces a pattern of antibody specificities characteristic of the anti-Sm and anti-RNP autoimmune response of patients with lupus erythematosus, as measured by monoclonal antibodies. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8689–8693. doi: 10.1073/pnas.83.22.8689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
  37. Ryder U., Sproat B. S., Lamond A. I. Sequence-specific affinity selection of mammalian splicing complexes. Nucleic Acids Res. 1990 Dec 25;18(24):7373–7379. doi: 10.1093/nar/18.24.7373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sass H., Pederson T. Transcription-dependent localization of U1 and U2 small nuclear ribonucleoproteins at major sites of gene activity in polytene chromosomes. J Mol Biol. 1984 Dec 25;180(4):911–926. doi: 10.1016/0022-2836(84)90263-8. [DOI] [PubMed] [Google Scholar]
  39. Scheer U., Benavente R. Functional and dynamic aspects of the mammalian nucleolus. Bioessays. 1990 Jan;12(1):14–21. doi: 10.1002/bies.950120104. [DOI] [PubMed] [Google Scholar]
  40. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  41. Smith H. C., Spector D. L., Woodcock C. L., Ochs R. L., Bhorjee J. Alterations in chromatin conformation are accompanied by reorganization of nonchromatin domains that contain U-snRNP protein p28 and nuclear protein p107. J Cell Biol. 1985 Aug;101(2):560–567. doi: 10.1083/jcb.101.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Smith J. H., Eliceiri G. L. Antibodies elicited by N2,N2-7-trimethylguanosine react with small nuclear RNAs and ribonucleoproteins. J Biol Chem. 1983 Apr 10;258(7):4636–4640. [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Spector D. L., Watt R. A., Sullivan N. F. The v- and c-myc oncogene proteins colocalize in situ with small nuclear ribonucleoprotein particles. Oncogene. 1987 Mar;1(1):5–12. [PubMed] [Google Scholar]
  46. Sproat B. S., Lamond A. I., Beijer B., Neuner P., Ryder U. Highly efficient chemical synthesis of 2'-O-methyloligoribonucleotides and tetrabiotinylated derivatives; novel probes that are resistant to degradation by RNA or DNA specific nucleases. Nucleic Acids Res. 1989 May 11;17(9):3373–3386. doi: 10.1093/nar/17.9.3373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Williams D. G., Stocks M. R., Smith P. R., Maini R. N. Murine lupus monoclonal antibodies define five epitopes on two different Sm polypeptides. Immunology. 1986 Jul;58(3):495–500. [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Zamore P. D., Green M. R. Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9243–9247. doi: 10.1073/pnas.86.23.9243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zieve G. W., Sauterer R. A. Cell biology of the snRNP particles. Crit Rev Biochem Mol Biol. 1990;25(1):1–46. doi: 10.3109/10409239009090604. [DOI] [PubMed] [Google Scholar]

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