Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 Mar 1;88(3):554–563. doi: 10.1083/jcb.88.3.554

hnRNA and its attachment to a nuclear protein matrix

PMCID: PMC2112768  PMID: 7217204

Abstract

In this study, DNA-depleted nuclear protein matrices are isolated from HeLa S3 cells. These nuclear matrices consist of peripheral laminae, residual nucleoli, and internal fibrillar structures. High molecular weight, heterogeneous nuclear RNA (hnRNA) is quantitatively associated with these structures and can be released intact only by affecting the integrity of the matrices. It is, therefore, concluded that hnRNA is part of a highly organized nuclear structure. By irradiation of intact cells or isolated nuclear matrices with ultraviolet light, proteins tightly associated with hnRNA can be induced to cross-link with the RNA. Performing the cross-linking in vivo is an extra guarantee that only hnRNA-protein (hnRNP) complexes existing in the intact cell are covalently linked. Such hnRNP complexes were isolated and purified under conditions that completely dissociate nonspecific RNA-protein complexes. By comparison of the hnRNP found in nuclear matrices and the published data on the composition of hnRNP particles, it was found that the so-called hnRNP "packaging" proteins (32,000-38,000 mol wt) were not efficiently cross-linked to hnRNA by UV irradiation. They were, however, present in the matrix preparations, bound to hnRNA, because they were released from nuclear matrices after ribonuclease treatment of these structures. On the other hand, two major hnRNPs (41,500 and 43,000 mol wt) were efficiently cross-linked to hnRNA. These proteins were not released by ribonuclease treatment, which suggests that they are involved in the binding of hnRNA to the nuclear matrix.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Aaronson R. P., Blobel G. Isolation of nuclear pore complexes in association with a lamina. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1007–1011. doi: 10.1073/pnas.72.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agutter P. S., Richardson J. C. Nuclear non-chromatin proteinaceous structures: their role in the organization and function of the interphase nucleus. J Cell Sci. 1980 Aug;44:395–435. doi: 10.1242/jcs.44.1.395. [DOI] [PubMed] [Google Scholar]
  3. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barrack E. R., Coffey D. S. The specific binding of estrogens and androgens to the nuclear matrix of sex hormone responsive tissues. J Biol Chem. 1980 Aug 10;255(15):7265–7275. [PubMed] [Google Scholar]
  5. Berezney R., Coffey D. S. Identification of a nuclear protein matrix. Biochem Biophys Res Commun. 1974 Oct 23;60(4):1410–1417. doi: 10.1016/0006-291x(74)90355-6. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Budzik G. P., Lam S. S., Schoemaker H. J., Schimmel P. R. Two photo-cross-linked complexes of isoleucine specific transfer ribonucleic acid with aminoacyl transfer ribonucleic acid synthetases. J Biol Chem. 1975 Jun 25;250(12):4433–4439. [PubMed] [Google Scholar]
  8. Comings D. E., Okada T. A. Nuclear proteins. III. The fibrillar nature of the nuclear matrix. Exp Cell Res. 1976 Dec;103(2):341–360. doi: 10.1016/0014-4827(76)90271-8. [DOI] [PubMed] [Google Scholar]
  9. Dubroff L. M., Nemer M. Molecular classes of heterogeneous nuclear RNA in sea urchin embryos. J Mol Biol. 1975 Jul 5;95(3):455–476. doi: 10.1016/0022-2836(75)90203-x. [DOI] [PubMed] [Google Scholar]
  10. Dwyer N., Blobel G. A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei. J Cell Biol. 1976 Sep;70(3):581–591. doi: 10.1083/jcb.70.3.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ehresmann B., Reinbolt J., Backendorf C., Tritsch D., Ebel J. Studies of the binding sites of Escherichia coli ribosomal protein S7 with 16S RNA by ultraviolet irradiation. FEBS Lett. 1976 Sep 1;67(3):316–319. doi: 10.1016/0014-5793(76)80555-8. [DOI] [PubMed] [Google Scholar]
  12. Ehresmann B., Reinbolt J., Ebel J. P. Studies of the binding of E. coli ribosomal protein S4 to 16S rRNA after UV irradiation of the S4--16S rRNA complex. FEBS Lett. 1975 Oct 15;58(1):106–111. doi: 10.1016/0014-5793(75)80236-5. [DOI] [PubMed] [Google Scholar]
  13. Faiferman I., Pogo A. O. Isolation of a nuclear ribonucleoprotein network that contains heterogeneous RNA and is bound to the nuclear envelope. Biochemistry. 1975 Aug 26;14(17):3808–3816. doi: 10.1021/bi00688a013. [DOI] [PubMed] [Google Scholar]
  14. Flytzanis C., Alonso A., Louis C., Krieg L., Sekeris C. E. Association of small nuclear RNA with HnRNA isolated from nuclear RNP complexes carrying HnRNA. FEBS Lett. 1978 Dec 1;96(1):201–206. doi: 10.1016/0014-5793(78)81094-1. [DOI] [PubMed] [Google Scholar]
  15. Franke W. W. Structure and function of nuclear membranes. Biochem Soc Symp. 1977;(42):125–135. [PubMed] [Google Scholar]
  16. Gerace L., Blum A., Blobel G. Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution. J Cell Biol. 1978 Nov;79(2 Pt 1):546–566. doi: 10.1083/jcb.79.2.546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Greenberg J. R. Ultraviolet light-induced crosslinking of mRNA to proteins. Nucleic Acids Res. 1979 Feb;6(2):715–732. doi: 10.1093/nar/6.2.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heinrich P. C., Gross V., Northemann W., Scheurlen M. Structure and function of nuclear ribonucleoprotein complexes. Rev Physiol Biochem Pharmacol. 1978;81:101–134. doi: 10.1007/BFb0034092. [DOI] [PubMed] [Google Scholar]
  19. Herlan G., Eckert W. A., Kaffenberger W., Wunderlich F. Isolation and characterization of an RNA-containing nuclear matrix from Tetrahymena macronuclei. Biochemistry. 1979 May 1;18(9):1782–1788. doi: 10.1021/bi00576a023. [DOI] [PubMed] [Google Scholar]
  20. Herman R., Weymouth L., Penman S. Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei. J Cell Biol. 1978 Sep;78(3):663–674. doi: 10.1083/jcb.78.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hodge L. D., Mancini P., Davis F. M., Heywood P. Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle. J Cell Biol. 1977 Jan;72(1):194–208. doi: 10.1083/jcb.72.1.194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Karn J., Vidali G., Boffa L. C., Allfrey V. G. Characterization of the non-histone nuclear proteins associated with rapidly labeled heterogeneous nuclear RNA. J Biol Chem. 1977 Oct 25;252(20):7307–7322. [PubMed] [Google Scholar]
  23. Krohne G., Franke W. W., Ely S., D'Arcy A., Jost E. Localization of a nuclear envelope-associated protein by indirect immunofluorescence microscopy using antibodies against a major polypeptide from rat liver fractions enriched in nuclear envelope-associated material. Cytobiologie. 1978 Oct;18(1):22–38. [PubMed] [Google Scholar]
  24. Kumar A., Pederson T. Comparison of proteins bound to heterogeneous nuclear RNA and messenger RNA in HeLa cells. J Mol Biol. 1975 Aug 15;96(3):353–365. doi: 10.1016/0022-2836(75)90165-5. [DOI] [PubMed] [Google Scholar]
  25. Long B. H., Huang C. Y., Pogo A. O. Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells: chromatin and hnRNA interactions with the nuclear matrix. Cell. 1979 Dec;18(4):1079–1090. doi: 10.1016/0092-8674(79)90221-6. [DOI] [PubMed] [Google Scholar]
  26. Louis C., Sekeris C. E. Isolation of informoferes from rat liver. Effects of alpha-amanitin and actinomycin D. Exp Cell Res. 1976 Oct 15;102(2):317–328. doi: 10.1016/0014-4827(76)90047-1. [DOI] [PubMed] [Google Scholar]
  27. Martinson H. G., Shetlar M. D., McCarthy B. J. Histone-histone interactions within chromatin. Crosslinking studies using ultraviolet light. Biochemistry. 1976 May 4;15(9):2002–2007. doi: 10.1021/bi00654a030. [DOI] [PubMed] [Google Scholar]
  28. Miller T. E., Huang C. Y., Pogo A. O. Rat liver nuclear skeleton and ribonucleoprotein complexes containing HnRNA. J Cell Biol. 1978 Mar;76(3):675–691. doi: 10.1083/jcb.76.3.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Miller T. E., Huang C. Y., Pogo A. O. Rat liver nuclear skeleton and small molecular weight RNA species. J Cell Biol. 1978 Mar;76(3):692–704. doi: 10.1083/jcb.76.3.692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mitchelson K. R., Bekers A. G., Wanka F. Isolation of a residual protein structure from nuclei of the myxomycete Physarum polycephalum. J Cell Sci. 1979 Oct;39:247–256. doi: 10.1242/jcs.39.1.247. [DOI] [PubMed] [Google Scholar]
  31. Pardoll D. M., Vogelstein B., Coffey D. S. A fixed site of DNA replication in eucaryotic cells. Cell. 1980 Feb;19(2):527–536. doi: 10.1016/0092-8674(80)90527-9. [DOI] [PubMed] [Google Scholar]
  32. Rosa J. J., Rosa M. D., Sigler P. B. Photocross-linking analysis of the contact surface of tRNA Met in complexes with Escherichia coli methionine:tRNA ligase. Biochemistry. 1979 Feb 20;18(4):637–647. doi: 10.1021/bi00571a014. [DOI] [PubMed] [Google Scholar]
  33. Samarina O. P., Lukanidin E. M., Molnar J., Georgiev G. P. Structural organization of nuclear complexes containing DNA-like RNA. J Mol Biol. 1968 Apr 14;33(1):251–263. doi: 10.1016/0022-2836(68)90292-1. [DOI] [PubMed] [Google Scholar]
  34. Schoemaker H. J., Schimmel P. R. Photo-induced joining of a transfer RNA with its cognate aminoacyl-transfer RNA synthetase. J Mol Biol. 1974 Apr 25;84(4):503–513. doi: 10.1016/0022-2836(74)90112-0. [DOI] [PubMed] [Google Scholar]
  35. Sekeris C. E., Niessing J. Evidence for the existence of a structural RNA component in the nuclear ribonucleoprotein particles containing heterogeneous RNA. Biochem Biophys Res Commun. 1975 Feb 3;62(3):642–650. doi: 10.1016/0006-291x(75)90447-7. [DOI] [PubMed] [Google Scholar]
  36. Smith G. K., Schray K. J., Schaffer S. W. Use of 5'-UTP-agarose for ribonuclease affinity chromatography. Anal Biochem. 1978 Feb;84(2):406–414. doi: 10.1016/0003-2697(78)90058-1. [DOI] [PubMed] [Google Scholar]
  37. Strniste G. F., Smith D. A. Induction of stable linkage between the deoxyribonucleic acid dependent ribonucleic acid polymerase and d(A-T)n-d(A-T)n by ultraviolet light. Biochemistry. 1974 Jan 29;13(3):485–493. doi: 10.1021/bi00700a014. [DOI] [PubMed] [Google Scholar]
  38. Wunderlich F., Herlan G. Reversibly contractile nuclear matrix. Its isolation, structure, and composition. J Cell Biol. 1977 May;73(2):271–278. doi: 10.1083/jcb.73.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES