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. 1992 Dec 1;119(5):1037–1046. doi: 10.1083/jcb.119.5.1037

Assembly and localization of the U1-specific snRNP C protein in the amphibian oocyte

PMCID: PMC2289723  PMID: 1447287

Abstract

To study the intranuclear localization of the U1-specific snRNP C protein and its assembly into U1 snRNPs, we injected transcripts encoding a myc-tagged C protein into amphibian oocytes. The distribution of protein translated from the injected RNA was essentially the same in continuous and pulse-label experiments. In both cases the C protein localized within the germinal vesicle in those structures known to contain U1 snRNPs, namely the lampbrush chromosome loops and hundreds of extrachromosomal granules called snurposomes. Oocytes were also injected with an antisense oligodeoxynucleotide that caused truncation of U1 snRNA at the 5' end. In these oocytes, myc- tagged C protein localized normally in the germinal vesicle and could be immunoprecipitated together with truncated U1 snRNA. These experiments suggest that the C protein can enter the germinal vesicle on its own and there associate with previously assembled U1 snRNPs. In transfected tissue culture cells, the myc-tagged C protein localized within the nucleus in a speckled pattern similar to that of endogenous U1 snRNPs.

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Selected References

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  1. Andersen J., Zieve G. W. Assembly and intracellular transport of snRNP particles. Bioessays. 1991 Feb;13(2):57–64. doi: 10.1002/bies.950130203. [DOI] [PubMed] [Google Scholar]
  2. Berg J. M. Proposed structure for the zinc-binding domains from transcription factor IIIA and related proteins. Proc Natl Acad Sci U S A. 1988 Jan;85(1):99–102. doi: 10.1073/pnas.85.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bringmann P., Lührmann R. Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents. EMBO J. 1986 Dec 20;5(13):3509–3516. doi: 10.1002/j.1460-2075.1986.tb04676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brody E., Abelson J. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction. Science. 1985 May 24;228(4702):963–967. doi: 10.1126/science.3890181. [DOI] [PubMed] [Google Scholar]
  6. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  7. Callan H. G., Gall J. G. Association of RNA with the B and C snurposomes of Xenopus oocyte nuclei. Chromosoma. 1991 Nov;101(2):69–82. doi: 10.1007/BF00357056. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Carter K. C., Taneja K. L., Lawrence J. B. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol. 1991 Dec;115(5):1191–1202. doi: 10.1083/jcb.115.5.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  13. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feeney R. J., Sauterer R. A., Feeney J. L., Zieve G. W. Cytoplasmic assembly and nuclear accumulation of mature small nuclear ribonucleoprotein particles. J Biol Chem. 1989 Apr 5;264(10):5776–5783. [PubMed] [Google Scholar]
  15. Frendewey D., Keller W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell. 1985 Aug;42(1):355–367. doi: 10.1016/s0092-8674(85)80131-8. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  19. Hamm J., Darzynkiewicz E., Tahara S. M., Mattaj I. W. The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal. Cell. 1990 Aug 10;62(3):569–577. doi: 10.1016/0092-8674(90)90021-6. [DOI] [PubMed] [Google Scholar]
  20. Hamm J., Dathan N. A., Mattaj I. W. Functional analysis of mutant Xenopus U2 snRNAs. Cell. 1989 Oct 6;59(1):159–169. doi: 10.1016/0092-8674(89)90878-7. [DOI] [PubMed] [Google Scholar]
  21. Krämer A., Utans U. Three protein factors (SF1, SF3 and U2AF) function in pre-splicing complex formation in addition to snRNPs. EMBO J. 1991 Jun;10(6):1503–1509. doi: 10.1002/j.1460-2075.1991.tb07670.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Lelay-Taha M. N., Reveillaud I., Sri-Widada J., Brunel C., Jeanteur P. RNA-protein organization of U1, U5 and U4-U6 small nuclear ribonucleoproteins in HeLa cells. J Mol Biol. 1986 Jun 5;189(3):519–532. doi: 10.1016/0022-2836(86)90321-9. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Nelissen R. L., Heinrichs V., Habets W. J., Simons F., Lührmann R., van Venrooij W. J. Zinc finger-like structure in U1-specific protein C is essential for specific binding to U1 snRNP. Nucleic Acids Res. 1991 Feb 11;19(3):449–454. doi: 10.1093/nar/19.3.449. [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. Osheim Y. N., Miller O. L., Jr, Beyer A. L. RNP particles at splice junction sequences on Drosophila chorion transcripts. Cell. 1985 Nov;43(1):143–151. doi: 10.1016/0092-8674(85)90019-4. [DOI] [PubMed] [Google Scholar]
  29. Paine P. L., Moore L. C., Horowitz S. B. Nuclear envelope permeability. Nature. 1975 Mar 13;254(5496):109–114. doi: 10.1038/254109a0. [DOI] [PubMed] [Google Scholar]
  30. Pan Z. Q., Prives C. Assembly of functional U1 and U2 human-amphibian hybrid snRNPs in Xenopus laevis oocytes. Science. 1988 Sep 9;241(4871):1328–1331. doi: 10.1126/science.2970672. [DOI] [PubMed] [Google Scholar]
  31. Pan Z. Q., Prives C. U2 snRNA sequences that bind U2-specific proteins are dispensable for the function of U2 snRNP in splicing. Genes Dev. 1989 Dec;3(12A):1887–1898. doi: 10.1101/gad.3.12a.1887. [DOI] [PubMed] [Google Scholar]
  32. Peculis B. A., Gall J. G. Localization of the nucleolar protein NO38 in amphibian oocytes. J Cell Biol. 1992 Jan;116(1):1–14. doi: 10.1083/jcb.116.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Prives C., Foukai D. Use of oligonucleotides for antisense experiments in Xenopus laevis oocytes. Methods Cell Biol. 1991;36:185–210. doi: 10.1016/s0091-679x(08)60278-2. [DOI] [PubMed] [Google Scholar]
  34. Roth M. B., Gall J. G. Targeting of a chromosomal protein to the nucleus and to lampbrush chromosome loops. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1269–1272. doi: 10.1073/pnas.86.4.1269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roth M. B., Zahler A. M., Stolk J. A. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. J Cell Biol. 1991 Nov;115(3):587–596. doi: 10.1083/jcb.115.3.587. [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. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [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. Sillekens P. T., Beijer R. P., Habets W. J., van Venrooij W. J. Human U1 snRNP-specific C protein: complete cDNA and protein sequence and identification of a multigene family in mammals. Nucleic Acids Res. 1988 Sep 12;16(17):8307–8321. doi: 10.1093/nar/16.17.8307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Steitz J. A. Immunoprecipitation of ribonucleoproteins using autoantibodies. Methods Enzymol. 1989;180:468–481. doi: 10.1016/0076-6879(89)80118-1. [DOI] [PubMed] [Google Scholar]
  43. Tafuri S. R., Wolffe A. P. Xenopus Y-box transcription factors: molecular cloning, functional analysis and developmental regulation. Proc Natl Acad Sci U S A. 1990 Nov;87(22):9028–9032. doi: 10.1073/pnas.87.22.9028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tsvetkov A., Jantsch M., Wu Z., Murphy C., Gall J. G. Transcription on lampbrush chromosome loops in the absence of U2 snRNA. Mol Biol Cell. 1992 Mar;3(3):249–261. doi: 10.1091/mbc.3.3.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Umesono K., Murakami K. K., Thompson C. C., Evans R. M. Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell. 1991 Jun 28;65(7):1255–1266. doi: 10.1016/0092-8674(91)90020-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wallace R. A., Jared D. W., Dumont J. N., Sega M. W. Protein incorporation by isolated amphibian oocytes. 3. Optimum incubation conditions. J Exp Zool. 1973 Jun;184(3):321–333. doi: 10.1002/jez.1401840305. [DOI] [PubMed] [Google Scholar]
  47. Wu Z. A., Murphy C., Callan H. G., Gall J. G. Small nuclear ribonucleoproteins and heterogeneous nuclear ribonucleoproteins in the amphibian germinal vesicle: loops, spheres, and snurposomes. J Cell Biol. 1991 May;113(3):465–483. doi: 10.1083/jcb.113.3.465. [DOI] [PMC free article] [PubMed] [Google Scholar]

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