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. 1988 Dec;85(24):9669–9673. doi: 10.1073/pnas.85.24.9669

Isolation and characterization of a Xenopus laevis C protein cDNA: structure and expression of a heterogeneous nuclear ribonucleoprotein core protein.

F Preugschat 1, B Wold 1
PMCID: PMC282832  PMID: 2904678

Abstract

The C proteins are major components of heterogeneous nuclear ribonucleoprotein complexes in nuclei of vertebrate cells. To begin to describe their structure, expression, and function we isolated and determined the DNA sequence of Xenopus laevis C protein cDNA clones. The protein predicted from the DNA sequence has a molecular mass of 30,916 kDa and is very similar to its human counterpart. Although mammalian genomes contain many copies of C protein sequence, the Xenopus genome contains few copies. When C protein RNA was synthesized in vitro and microinjected into stage-VI Xenopus oocytes, newly synthesized C proteins were efficiently localized in the nucleus. In vitro rabbit reticulocyte lysate and in vivo Xenopus oocyte translation systems both produce from a single mRNA two discrete polypeptide species that accumulate in a ratio similar to that of mammalian C1 and C2 proteins in vivo.

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Selected References

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

  1. Adam S. A., Nakagawa T., Swanson M. S., Woodruff T. K., Dreyfuss G. mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol Cell Biol. 1986 Aug;6(8):2932–2943. doi: 10.1128/mcb.6.8.2932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adeniyi-Jones S., Zasloff M. Transcription, processing and nuclear transport of a B1 Alu RNA species complementary to an intron of the murine alpha-fetoprotein gene. Nature. 1985 Sep 5;317(6032):81–84. doi: 10.1038/317081a0. [DOI] [PubMed] [Google Scholar]
  3. Beyer A. L., Bouton A. H., Miller O. L., Jr Correlation of hnRNP structure and nascent transcript cleavage. Cell. 1981 Oct;26(2 Pt 2):155–165. doi: 10.1016/0092-8674(81)90299-3. [DOI] [PubMed] [Google Scholar]
  4. Beyer A. L., Christensen M. E., Walker B. W., LeStourgeon W. M. Identification and characterization of the packaging proteins of core 40S hnRNP particles. Cell. 1977 May;11(1):127–138. doi: 10.1016/0092-8674(77)90323-3. [DOI] [PubMed] [Google Scholar]
  5. Buvoli M., Biamonti G., Tsoulfas P., Bassi M. T., Ghetti A., Riva S., Morandi C. cDNA cloning of human hnRNP protein A1 reveals the existence of multiple mRNA isoforms. Nucleic Acids Res. 1988 May 11;16(9):3751–3770. doi: 10.1093/nar/16.9.3751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  7. Choi Y. D., Dreyfuss G. Isolation of the heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): a unique supramolecular assembly. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7471–7475. doi: 10.1073/pnas.81.23.7471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Choi Y. D., Grabowski P. J., Sharp P. A., Dreyfuss G. Heterogeneous nuclear ribonucleoproteins: role in RNA splicing. Science. 1986 Mar 28;231(4745):1534–1539. doi: 10.1126/science.3952495. [DOI] [PubMed] [Google Scholar]
  9. Chung S. Y., Wooley J. Set of novel, conserved proteins fold pre-messenger RNA into ribonucleosomes. Proteins. 1986 Nov;1(3):195–210. doi: 10.1002/prot.340010302. [DOI] [PubMed] [Google Scholar]
  10. Cobianchi F., SenGupta D. N., Zmudzka B. Z., Wilson S. H. Structure of rodent helix-destabilizing protein revealed by cDNA cloning. J Biol Chem. 1986 Mar 15;261(8):3536–3543. [PubMed] [Google Scholar]
  11. Conway L., Wickens M. A sequence downstream of A-A-U-A-A-A is required for formation of simian virus 40 late mRNA 3' termini in frog oocytes. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3949–3953. doi: 10.1073/pnas.82.12.3949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. De Robertis E. M., Black P., Nishikura K. Intranuclear location of the tRNA splicing enzymes. Cell. 1981 Jan;23(1):89–93. doi: 10.1016/0092-8674(81)90273-7. [DOI] [PubMed] [Google Scholar]
  13. Dingwall C., Dilworth S. M., Black S. J., Kearsey S. E., Cox L. S., Laskey R. A. Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals. EMBO J. 1987 Jan;6(1):69–74. doi: 10.1002/j.1460-2075.1987.tb04720.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dreyfuss G., Choi Y. D., Adam S. A. Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies. Mol Cell Biol. 1984 Jun;4(6):1104–1114. doi: 10.1128/mcb.4.6.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dreyfuss G., Choi Y. D., Adam S. A. Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies. Mol Cell Biol. 1984 Jun;4(6):1104–1114. doi: 10.1128/mcb.4.6.1104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dreyfuss G. Structure and function of nuclear and cytoplasmic ribonucleoprotein particles. Annu Rev Cell Biol. 1986;2:459–498. doi: 10.1146/annurev.cb.02.110186.002331. [DOI] [PubMed] [Google Scholar]
  17. Earnshaw W. C., Honda B. M., Laskey R. A., Thomas J. O. Assembly of nucleosomes: the reaction involving X. laevis nucleoplasmin. Cell. 1980 Sep;21(2):373–383. doi: 10.1016/0092-8674(80)90474-2. [DOI] [PubMed] [Google Scholar]
  18. Fitzgerald M., Shenk T. The sequence 5'-AAUAAA-3'forms parts of the recognition site for polyadenylation of late SV40 mRNAs. Cell. 1981 Apr;24(1):251–260. doi: 10.1016/0092-8674(81)90521-3. [DOI] [PubMed] [Google Scholar]
  19. Foe V. E., Wilkinson L. E., Laird C. D. Comparative organization of active transcription units in Oncopeltus fasciatus. Cell. 1976 Sep;9(1):131–146. doi: 10.1016/0092-8674(76)90059-3. [DOI] [PubMed] [Google Scholar]
  20. Galli G., Hofstetter H., Stunnenberg H. G., Birnstiel M. L. Biochemical complementation with RNA in the Xenopus oocyte: a small RNA is required for the generation of 3' histone mRNA termini. Cell. 1983 Oct;34(3):823–828. doi: 10.1016/0092-8674(83)90539-1. [DOI] [PubMed] [Google Scholar]
  21. Georgiev O., Mous J., Birnstiel M. L. Processing and nucleo-cytoplasmic transport of histone gene transcripts. Nucleic Acids Res. 1984 Nov 26;12(22):8539–8551. doi: 10.1093/nar/12.22.8539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grange T., de Sa C. M., Oddos J., Pictet R. Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif. Nucleic Acids Res. 1987 Jun 25;15(12):4771–4787. doi: 10.1093/nar/15.12.4771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Green M. R., Maniatis T., Melton D. A. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell. 1983 Mar;32(3):681–694. doi: 10.1016/0092-8674(83)90054-5. [DOI] [PubMed] [Google Scholar]
  24. Habets W. J., Sillekens P. T., Hoet M. H., Schalken J. A., Roebroek A. J., Leunissen J. A., van de Ven W. J., van Venrooij W. J. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B" antigen. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2421–2425. doi: 10.1073/pnas.84.8.2421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hathaway G. M., Traugh J. A. Casein kinases--multipotential protein kinases. Curr Top Cell Regul. 1982;21:101–127. [PubMed] [Google Scholar]
  26. Hattori M., Sakaki Y. Dideoxy sequencing method using denatured plasmid templates. Anal Biochem. 1986 Feb 1;152(2):232–238. doi: 10.1016/0003-2697(86)90403-3. [DOI] [PubMed] [Google Scholar]
  27. Haynes S. R., Rebbert M. L., Mozer B. A., Forquignon F., Dawid I. B. pen repeat sequences are GGN clusters and encode a glycine-rich domain in a Drosophila cDNA homologous to the rat helix destabilizing protein. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1819–1823. doi: 10.1073/pnas.84.7.1819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Holcomb E. R., Friedman D. L. Phosphorylation of the C-proteins of HeLa cell hnRNP particles. Involvement of a casein kinase II-type enzyme. J Biol Chem. 1984 Jan 10;259(1):31–40. [PubMed] [Google Scholar]
  29. Jong A. Y., Clark M. W., Gilbert M., Oehm A., Campbell J. L. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins. Mol Cell Biol. 1987 Aug;7(8):2947–2955. doi: 10.1128/mcb.7.8.2947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kleinschmidt J. A., Fortkamp E., Krohne G., Zentgraf H., Franke W. W. Co-existence of two different types of soluble histone complexes in nuclei of Xenopus laevis oocytes. J Biol Chem. 1985 Jan 25;260(2):1166–1176. [PubMed] [Google Scholar]
  31. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Krieg P. A., Melton D. A. Formation of the 3' end of histone mRNA by post-transcriptional processing. Nature. 1984 Mar 8;308(5955):203–206. doi: 10.1038/308203a0. [DOI] [PubMed] [Google Scholar]
  33. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  34. Lahiri D. K., Thomas J. O. A cDNA clone of the hnRNP C proteins and its homology with the single-stranded DNA binding protein UP2. Nucleic Acids Res. 1986 May 27;14(10):4077–4094. doi: 10.1093/nar/14.10.4077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lapeyre B., Bourbon H., Amalric F. Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1472–1476. doi: 10.1073/pnas.84.6.1472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  37. Lothstein L., Arenstorf H. P., Chung S. Y., Walker B. W., Wooley J. C., LeStourgeon W. M. General organization of protein in HeLa 40S nuclear ribonucleoprotein particles. J Cell Biol. 1985 May;100(5):1570–1581. doi: 10.1083/jcb.100.5.1570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Malcolm D. B., Sommerville J. The structure of nuclear ribonucleoprotein of amphibian oocytes. J Cell Sci. 1977 Apr;24:143–165. doi: 10.1242/jcs.24.1.143. [DOI] [PubMed] [Google Scholar]
  39. Martin T., Billings P., Pullman J., Stevens B., Kinniburgh A. Substructure of nuclear ribonucleoprotein complexes. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):899–909. doi: 10.1101/sqb.1978.042.01.091. [DOI] [PubMed] [Google Scholar]
  40. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  41. Mayrand S., Pederson T. Nuclear ribonucleoprotein particles probed in living cells. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2208–2212. doi: 10.1073/pnas.78.4.2208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. McKnight S. L., Miller O. L., Jr Ultrastructural patterns of RNA synthesis during early embryogenesis of Drosophila melanogaster. Cell. 1976 Jun;8(2):305–319. doi: 10.1016/0092-8674(76)90014-3. [DOI] [PubMed] [Google Scholar]
  43. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Merrill B. M., LoPresti M. B., Stone K. L., Williams K. R. High pressure liquid chromatography purification of UP1 and UP2, two related single-stranded nucleic acid-binding proteins from calf thymus. J Biol Chem. 1986 Jan 15;261(2):878–883. [PubMed] [Google Scholar]
  45. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  46. Nakagawa T. Y., Swanson M. S., Wold B. J., Dreyfuss G. Molecular cloning of cDNA for the nuclear ribonucleoprotein particle C proteins: a conserved gene family. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2007–2011. doi: 10.1073/pnas.83.7.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pederson T. Proteins associated with heterogeneous nuclear RNA in eukaryotic cells. J Mol Biol. 1974 Feb 25;83(2):163–183. doi: 10.1016/0022-2836(74)90386-6. [DOI] [PubMed] [Google Scholar]
  48. Rebagliati M. R., Weeks D. L., Harvey R. P., Melton D. A. Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell. 1985 Oct;42(3):769–777. doi: 10.1016/0092-8674(85)90273-9. [DOI] [PubMed] [Google Scholar]
  49. Sachs A. B., Bond M. W., Kornberg R. D. A single gene from yeast for both nuclear and cytoplasmic polyadenylate-binding proteins: domain structure and expression. Cell. 1986 Jun 20;45(6):827–835. doi: 10.1016/0092-8674(86)90557-x. [DOI] [PubMed] [Google Scholar]
  50. Sachs A. B., Davis R. W., Kornberg R. D. A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Mol Cell Biol. 1987 Sep;7(9):3268–3276. doi: 10.1128/mcb.7.9.3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Samarina O. P., Molnar J., Lukanidin E. M., Bruskov V. I., Krichevskaya A. A., Georgiev G. P. Reversible dissociation of nuclear ribonucleoprotein particle containing mRNA into RNA and protein. J Mol Biol. 1967 Jul 14;27(1):187–191. doi: 10.1016/0022-2836(67)90359-2. [DOI] [PubMed] [Google Scholar]
  52. Setyono B., Greenberg J. R. Proteins associated with poly(A) and other regions of mRNA and hnRNA molecules as investigated by crosslinking. Cell. 1981 Jun;24(3):775–783. doi: 10.1016/0092-8674(81)90103-3. [DOI] [PubMed] [Google Scholar]
  53. Sillekens P. T., Habets W. J., Beijer R. P., van Venrooij W. J. cDNA cloning of the human U1 snRNA-associated A protein: extensive homology between U1 and U2 snRNP-specific proteins. EMBO J. 1987 Dec 1;6(12):3841–3848. doi: 10.1002/j.1460-2075.1987.tb02721.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Smith A. E., Kalderon D., Roberts B. L., Colledge W. H., Edge M., Gillett P., Markham A., Paucha E., Richardson W. D. The nuclear location signal. Proc R Soc Lond B Biol Sci. 1985 Oct 22;226(1242):43–58. doi: 10.1098/rspb.1985.0078. [DOI] [PubMed] [Google Scholar]
  55. Swanson M. S., Nakagawa T. Y., LeVan K., Dreyfuss G. Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins. Mol Cell Biol. 1987 May;7(5):1731–1739. doi: 10.1128/mcb.7.5.1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Theissen H., Etzerodt M., Reuter R., Schneider C., Lottspeich F., Argos P., Lührmann R., Philipson L. Cloning of the human cDNA for the U1 RNA-associated 70K protein. EMBO J. 1986 Dec 1;5(12):3209–3217. doi: 10.1002/j.1460-2075.1986.tb04631.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Van Eekelen C. A., Mariman E. C., Reinders R. J., Van Venrooij W. J. Adenoviral heterogeneous nuclear RNA is associated with host cell proteins. Eur J Biochem. 1981 Oct;119(3):461–467. doi: 10.1111/j.1432-1033.1981.tb05630.x. [DOI] [PubMed] [Google Scholar]
  58. Wilk H. E., Werr H., Friedrich D., Kiltz H. H., Schäfer K. P. The core proteins of 35S hnRNP complexes. Characterization of nine different species. Eur J Biochem. 1985 Jan 2;146(1):71–81. doi: 10.1111/j.1432-1033.1985.tb08621.x. [DOI] [PubMed] [Google Scholar]
  59. Zasloff M., Rosenberg M., Santos T. Impaired nuclear transport of a human variant tRNAiMet. Nature. 1982 Nov 4;300(5887):81–84. doi: 10.1038/300081a0. [DOI] [PubMed] [Google Scholar]
  60. Zasloff M. tRNA transport from the nucleus in a eukaryotic cell: carrier-mediated translocation process. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6436–6440. doi: 10.1073/pnas.80.21.6436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. de Vegvar H. E., Lund E., Dahlberg J. E. 3' end formation of U1 snRNA precursors is coupled to transcription from snRNA promoters. Cell. 1986 Oct 24;47(2):259–266. doi: 10.1016/0092-8674(86)90448-4. [DOI] [PubMed] [Google Scholar]
  62. van Eekelen C. A., Riemen T., van Venrooij W. J. Specificity in the interaction of hnRNA and mRNA with proteins as revealed by in vivo cross linking. FEBS Lett. 1981 Aug 3;130(2):223–226. doi: 10.1016/0014-5793(81)81125-8. [DOI] [PubMed] [Google Scholar]

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