Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1991 Jul;10(7):1863–1873. doi: 10.1002/j.1460-2075.1991.tb07712.x

In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells.

M Carmo-Fonseca 1, R Pepperkok 1, B S Sproat 1, W Ansorge 1, M S Swanson 1, A I Lamond 1
PMCID: PMC452861  PMID: 1710980

Abstract

The in vivo distribution of snRNPs has been analysed by microinjecting fluorochrome-labelled antisense probes into the nuclei of live HeLa and 3T3 cells. Probes for U2 and U5 snRNAs specifically label the same discrete nuclear foci while a probe for U1 snRNA shows widespread nucleoplasmic labelling, excluding nucleoli, in addition to labelling foci. A probe for U3 snRNA specifically labels nucleoli. These in vivo data confirm that mammalian cells have nuclear foci which contain spliceosomal snRNPs. Co-localization studies, both in vivo and in situ, demonstrate that the spliceosomal snRNAs are present in the same nuclear foci. These foci are also stained by antibodies which recognize snRNP proteins, m3G-cap structures and the splicing factor U2AF but are not stained by anti-SC-35 or anti-La antibodies. U1 snRNP and the splicing factor U2AF closely co-localize in the nucleus, both before and after actinomycin D treatment, suggesting that they may both be part of the same complex in vivo.

Full text

PDF
1863

Images in this article

1864Image
on p.1864
1865Image
on p.1865
1865Image
on p.1865
1866Image
on p.1866
1867Image
on p.1867
1868Image
on p.1868
1869Image
on p.1869
1870Image
on p.1870
1871Image
on p.1871

Selected References

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

  1. Ansorge W., Pepperkok R. Performance of an automated system for capillary microinjection into living cells. J Biochem Biophys Methods. 1988 Aug;16(4):283–292. doi: 10.1016/0165-022x(88)90062-0. [DOI] [PubMed] [Google Scholar]
  2. Bachmann M., Mayet W. J., Schröder H. C., Pfeifer K., Meyer zum Büschenfelde K. H., Müller W. E. Association of La and Ro antigens with intracellular structures in HEp-2 carcinoma cells. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7770–7774. doi: 10.1073/pnas.83.20.7770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bindereif A., Green M. R. Identification and functional analysis of mammalian splicing factors. Genet Eng (N Y) 1990;12:201–224. doi: 10.1007/978-1-4613-0641-2_11. [DOI] [PubMed] [Google Scholar]
  5. Bochnig P., Reuter R., Bringmann P., Lührmann R. A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7-methylguanosine-capped RNAs. Eur J Biochem. 1987 Oct 15;168(2):461–467. doi: 10.1111/j.1432-1033.1987.tb13439.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Company M., Arenas J., Abelson J. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature. 1991 Feb 7;349(6309):487–493. doi: 10.1038/349487a0. [DOI] [PubMed] [Google Scholar]
  9. Connolly B. A. The synthesis of oligonucleotides containing a primary amino group at the 5'-terminus. Nucleic Acids Res. 1987 Apr 10;15(7):3131–3139. doi: 10.1093/nar/15.7.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Jacquier A. Self-splicing group II and nuclear pre-mRNA introns: how similar are they? Trends Biochem Sci. 1990 Sep;15(9):351–354. doi: 10.1016/0968-0004(90)90075-m. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Padgett R. A., Konarska M. M., Grabowski P. J., Hardy S. F., Sharp P. A. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science. 1984 Aug 31;225(4665):898–903. doi: 10.1126/science.6206566. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Pieles U., Sproat B. S., Lamm G. M. A protected biotin containing deoxycytidine building block for solid phase synthesis of biotinylated oligonucleotides. Nucleic Acids Res. 1990 Aug 11;18(15):4355–4360. doi: 10.1093/nar/18.15.4355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Piñol-Roma S., Swanson M. S., Gall J. G., Dreyfuss G. A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts. J Cell Biol. 1989 Dec;109(6 Pt 1):2575–2587. doi: 10.1083/jcb.109.6.2575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Roget A., Bazin H., Teoule R. Synthesis and use of labelled nucleoside phosphoramidite building blocks bearing a reporter group: biotinyl, dinitrophenyl, pyrenyl and dansyl. Nucleic Acids Res. 1989 Oct 11;17(19):7643–7651. doi: 10.1093/nar/17.19.7643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Sproat B. S., Iribarren A. M., Garcia R. G., Beijer B. New synthetic routes to synthons suitable for 2'-O-allyloligoribonucleotide assembly. Nucleic Acids Res. 1991 Feb 25;19(4):733–738. doi: 10.1093/nar/19.4.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. 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]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES