Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 Dec 11;18(23):6989–6995. doi: 10.1093/nar/18.23.6989

A nuclear cap binding protein from HeLa cells.

M Ohno 1, N Kataoka 1, Y Shimura 1
PMCID: PMC332760  PMID: 2148205

Abstract

We have identified a cap binding protein in a HeLa nuclear extract using a gel mobility shift assay probed with capped RNA. Subcellular fractionation of HeLa cells revealed that the majority (about 70%) of the cap binding activity is present in the nuclear extract, about 20% is in the cytoplasmic S100 fraction, and almost none in the ribosome-high salt wash fraction, indicating that the protein in active form localizes mainly in the nuclei. Competition experiments with various cap analogues showed that the G(5')ppp(5')N-blocking structure as well as the methyl residue at the N7 position of the blocking guanosine is important for the binding of this protein, and that the trimethylguanosine cap structure which exists at the 5' termini of many snRNAs is not recognized by this protein. Immunoprecipitation experiments using various anti-snRNP antibodies suggested that this protein is partially associated with U2 snRNP. We purified this protein to near homogeneity from a HeLa nuclear extract by several chromatographic procedures including capped RNA-Sepharose chromatography. The purified protein shows molecular weight of 80 kilodaltons, as judged by SDS gel electrophoresis, and binds specifically to the cap structure.

Full text

PDF
6992

Images in this article

6991Image
on p.6991
6991Image
on p.6991
6992Image
on p.6992
6992Image
on p.6992
6993Image
on p.6993
6993Image
on p.6993

Selected References

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

  1. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Edery I., Sonenberg N. Cap-dependent RNA splicing in a HeLa nuclear extract. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7590–7594. doi: 10.1073/pnas.82.22.7590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hellmann G. M., Chu L. Y., Rhoads R. E. A polypeptide which reverses cap analogue inhibition of cell-free protein synthesis. Purification and binding to capped oligonucleotides. J Biol Chem. 1982 Apr 25;257(8):4056–4062. [PubMed] [Google Scholar]
  5. Inoue K., Ohno M., Sakamoto H., Shimura Y. Effect of the cap structure on pre-mRNA splicing in Xenopus oocyte nuclei. Genes Dev. 1989 Sep;3(9):1472–1479. doi: 10.1101/gad.3.9.1472. [DOI] [PubMed] [Google Scholar]
  6. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Konarska M. M., Padgett R. A., Sharp P. A. Recognition of cap structure in splicing in vitro of mRNA precursors. Cell. 1984 Oct;38(3):731–736. doi: 10.1016/0092-8674(84)90268-x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Mimori T., Hinterberger M., Pettersson I., Steitz J. A. Autoantibodies to the U2 small nuclear ribonucleoprotein in a patient with scleroderma-polymyositis overlap syndrome. J Biol Chem. 1984 Jan 10;259(1):560–565. [PubMed] [Google Scholar]
  12. Mizumoto K., Kaziro Y. Messenger RNA capping enzymes from eukaryotic cells. Prog Nucleic Acid Res Mol Biol. 1987;34:1–28. doi: 10.1016/s0079-6603(08)60491-2. [DOI] [PubMed] [Google Scholar]
  13. Ohno M., Sakamoto H., Shimura Y. Preferential excision of the 5' proximal intron from mRNA precursors with two introns as mediated by the cap structure. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5187–5191. doi: 10.1073/pnas.84.15.5187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Padgett R. A., Mount S. M., Steitz J. A., Sharp P. A. Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein. Cell. 1983 Nov;35(1):101–107. doi: 10.1016/0092-8674(83)90212-x. [DOI] [PubMed] [Google Scholar]
  15. Patzelt E., Blaas D., Kuechler E. CAP binding proteins associated with the nucleus. Nucleic Acids Res. 1983 Sep 10;11(17):5821–5835. doi: 10.1093/nar/11.17.5821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Patzelt E., Thalmann E., Hartmuth K., Blaas D., Kuechler E. Assembly of pre-mRNA splicing complex is cap dependent. Nucleic Acids Res. 1987 Feb 25;15(4):1387–1399. doi: 10.1093/nar/15.4.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rhoads R. E. Cap recognition and the entry of mRNA into the protein synthesis initiation cycle. Trends Biochem Sci. 1988 Feb;13(2):52–56. doi: 10.1016/0968-0004(88)90028-x. [DOI] [PubMed] [Google Scholar]
  18. Rozen F., Sonenberg N. Identification of nuclear cap specific proteins in HeLa cells. Nucleic Acids Res. 1987 Aug 25;15(16):6489–6500. doi: 10.1093/nar/15.16.6489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rychlik W., Domier L. L., Gardner P. R., Hellmann G. M., Rhoads R. E. Amino acid sequence of the mRNA cap-binding protein from human tissues. Proc Natl Acad Sci U S A. 1987 Feb;84(4):945–949. doi: 10.1073/pnas.84.4.945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sakamoto H., Ohno M., Yasuda K., Mizumoto K., Shimura Y. In vitro splicing of a chicken delta-crystallin pre-mRNA in a mammalian nuclear extract. J Biochem. 1987 Nov;102(5):1289–1301. doi: 10.1093/oxfordjournals.jbchem.a122167. [DOI] [PubMed] [Google Scholar]
  21. Shatkin A. J. mRNA cap binding proteins: essential factors for initiating translation. Cell. 1985 Feb;40(2):223–224. doi: 10.1016/0092-8674(85)90132-1. [DOI] [PubMed] [Google Scholar]
  22. Sonenberg N. ATP/Mg++-dependent cross-linking of cap binding proteins to the 5' end of eukaryotic mRNA. Nucleic Acids Res. 1981 Apr 10;9(7):1643–1656. doi: 10.1093/nar/9.7.1643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sonenberg N., Rupprecht K. M., Hecht S. M., Shatkin A. J. Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4345–4349. doi: 10.1073/pnas.76.9.4345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Trachsel H., Sonenberg N., Shatkin A. J., Rose J. K., Leong K., Bergmann J. E., Gordon J., Baltimore D. Purification of a factor that restores translation of vesicular stomatitis virus mRNA in extracts from poliovirus-infected HeLa cells. Proc Natl Acad Sci U S A. 1980 Feb;77(2):770–774. doi: 10.1073/pnas.77.2.770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Webb N. R., Chari R. V., DePillis G., Kozarich J. W., Rhoads R. E. Purification of the messenger RNA cap-binding protein using a new affinity medium. Biochemistry. 1984 Jan 17;23(2):177–181. doi: 10.1021/bi00297a001. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES