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. 1981 Aug 1;90(2):380–384. doi: 10.1083/jcb.90.2.380

Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells

PMCID: PMC2111869  PMID: 6169730

Abstract

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA- protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell.

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Selected References

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  1. 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]
  2. Beyer A. L., Miller O. L., Jr, McKnight S. L. Ribonucleoprotein structure in nascent hnRNA is nonrandom and sequence-dependent. Cell. 1980 May;20(1):75–84. doi: 10.1016/0092-8674(80)90236-6. [DOI] [PubMed] [Google Scholar]
  3. Bhorjee J. S., Pederson T. Chromosomal proteins: tightly bound nucleic acid and its bearing on the measurement of nonhistone protein phosphorylation. Anal Biochem. 1976 Apr;71(2):393–404. doi: 10.1016/s0003-2697(76)80005-x. [DOI] [PubMed] [Google Scholar]
  4. Bhorjee J. S., Pederson T. Nonhistone chromosomal proteins in synchronized HeLa cells. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3345–3349. doi: 10.1073/pnas.69.11.3345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Busby S., Bakken A. A quantitative electron microscopic analysis of transcription in sea urchin embryos. Chromosoma. 1979 Mar 12;71(3):249–262. doi: 10.1007/BF00287135. [DOI] [PubMed] [Google Scholar]
  6. Calvet J. P., Pederson T. Heterogeneous nuclear RNA double-stranded regions probed in living HeLa cells by crosslinking with the psoralen derivative aminomethyltrioxsalen. Proc Natl Acad Sci U S A. 1979 Feb;76(2):755–759. doi: 10.1073/pnas.76.2.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Calvet J. P., Pederson T. Nucleoprotein organization of inverted repeat DNA transcripts in heterogeneous nuclear RNA-ribonucleoprotein particles from HeLa cells. J Mol Biol. 1978 Jul 5;122(3):361–378. doi: 10.1016/0022-2836(78)90195-x. [DOI] [PubMed] [Google Scholar]
  8. Calvet J. P., Pederson T. Photochemical cross-linking of secondary structure in HeLa cell heterogeneous nuclear RNA in situ 1. Nucleic Acids Res. 1979;6(5):1993–2001. doi: 10.1093/nar/6.5.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Calvet J. P., Pederson T. Secondary structure of heterogeneous nuclear RNA: two classes of double-stranded RNA in native ribonucleoprotein. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3705–3709. doi: 10.1073/pnas.74.9.3705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Firtel R. A., Pederson T. Ribonucleoprotein particles containing heterogeneous nuclear RNA in the cellular slime mold Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1975 Jan;72(1):301–305. doi: 10.1073/pnas.72.1.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Ford J. P., Hsu M. T. Transcription pattern of in vivo-labeled late simian virus 40 RNA: equimolar transcription beyond the mRNA 3' terminus. J Virol. 1978 Dec;28(3):795–801. doi: 10.1128/jvi.28.3.795-801.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. GALL J. G. On the submicroscopic structure of chromosomes. Brookhaven Symp Biol. 1956 Feb;(8):17–32. [PubMed] [Google Scholar]
  14. Greenberg J. R., Perry R. P. Relative occurrence of polyadenylic acid sequences in messenger and heterogeneous nuclear RNA of L cells as determined by poly (U)-hydroxylapatite chromatography. J Mol Biol. 1972 Dec 14;72(1):91–98. doi: 10.1016/0022-2836(72)90070-8. [DOI] [PubMed] [Google Scholar]
  15. Greenberg J. R. Proteins crosslinked to messenger RNA by irradiating polyribosomes with ultraviolet light. Nucleic Acids Res. 1980 Dec 11;8(23):5685–5701. doi: 10.1093/nar/8.23.5685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Hofer E., Darnell J. E., Jr The primary transcription unit of the mouse beta-major globin gene. Cell. 1981 Feb;23(2):585–593. doi: 10.1016/0092-8674(81)90154-9. [DOI] [PubMed] [Google Scholar]
  18. Holland C. A., Mayrand S., Pederson T. Sequence complexity of nuclear and messenger RNA in HeLa cells. J Mol Biol. 1980 Apr 25;138(4):755–778. doi: 10.1016/0022-2836(80)90064-9. [DOI] [PubMed] [Google Scholar]
  19. Jelinek W., Adesnik M., Salditt M., Sheiness D., Wall R., Molloy G., Philipson L., Darnell J. E. Further evidence on the nuclear origin and transfer to the cytoplasm of polyadenylic acid sequences in mammalian cell RNA. J Mol Biol. 1973 Apr 15;75(3):515–532. doi: 10.1016/0022-2836(73)90458-0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kinniburgh A. J., Martin T. E. Detection of mRNA sequences in nuclear 30S ribonucleoprotein subcomplexes. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2725–2729. doi: 10.1073/pnas.73.8.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kish V. M., Pederson T. Heterogeneous nuclear RNA secondary structure: oligo (U) sequences base-paired with poly (A) and their possible role as binding sites for heterogeneous nuclear RNA-specific proteins. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1426–1430. doi: 10.1073/pnas.74.4.1426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kish V. M., Pederson T. Isolation and characterization of ribonucleoprotein particles containing heterogeneous nuclear RNA. Methods Cell Biol. 1978;17:377–399. doi: 10.1016/s0091-679x(08)61155-3. [DOI] [PubMed] [Google Scholar]
  24. Kish V. M., Pederson T. Ribonucleoprotein organization of polyadenylate sequences in HeLa cell heterogeneous nuclear RNA. J Mol Biol. 1975 Jun 25;95(2):227–238. doi: 10.1016/0022-2836(75)90392-7. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Kumar A., Warner J. R. Characterization of ribosomal precursor particles from HeLa cell nucleoli. J Mol Biol. 1972 Jan 28;63(2):233–246. doi: 10.1016/0022-2836(72)90372-5. [DOI] [PubMed] [Google Scholar]
  27. Laird C. D., Chooi W. Y. Morphology of transcription units in Drosophila melanogaster. Chromosoma. 1976 Oct 28;58(2):193–218. doi: 10.1007/BF00701359. [DOI] [PubMed] [Google Scholar]
  28. Laird C. D., Wilkinson L. E., Foe V. E., Chooi W. Y. Analysis of chromatin-associated fiber arrays. Chromosoma. 1976 Oct 28;58(2):169–190. doi: 10.1007/BF00701357. [DOI] [PubMed] [Google Scholar]
  29. Lamb M. M., Daneholt B. Characterization of active transcription units in Balbiani rings of Chironomus tentans. Cell. 1979 Aug;17(4):835–848. doi: 10.1016/0092-8674(79)90324-6. [DOI] [PubMed] [Google Scholar]
  30. Malcolm D. B., Sommerville J. The structure of chromosome-derived ribonucleoprotein in oocytes of Triturus cristatus carnifex (Laurenti). Chromosoma. 1974;48(2):137–158. doi: 10.1007/BF00283960. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. McKnight S. L., Miller O. L., Jr Post-replicative nonribosomal transcription units in D. melanogaster embryos. Cell. 1979 Jul;17(3):551–563. doi: 10.1016/0092-8674(79)90263-0. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Monneron A., Bernhard W. Fine structural organization of the interphase nucleus in some mammalian cells. J Ultrastruct Res. 1969 May;27(3):266–288. doi: 10.1016/s0022-5320(69)80017-1. [DOI] [PubMed] [Google Scholar]
  35. Munroe S. H., Pederson T. Messenger RNA sequences in nuclear ribonucleoprotein particles are complexed with protein as shown by nuclease protection. J Mol Biol. 1981 Apr 15;147(3):437–449. doi: 10.1016/0022-2836(81)90494-0. [DOI] [PubMed] [Google Scholar]
  36. Möller K., Brimacombe R. Specific cross-linking of proteins S7 and L4 to ribosomal RNA, by UV irradiation of Escherichia coli ribosomal subunits. Mol Gen Genet. 1975 Dec 9;141(4):343–355. doi: 10.1007/BF00331455. [DOI] [PubMed] [Google Scholar]
  37. Pederson T., Bhorjee J. S. Evidence for a role of RNA in eukaryotic chromosome structure. Metabolically stable, small nuclear RNA species are covalently linked to chromosomal DNA in HeLa cells. J Mol Biol. 1979 Mar 15;128(4):451–480. doi: 10.1016/0022-2836(79)90288-2. [DOI] [PubMed] [Google Scholar]
  38. Pederson T. Chromatin structure and the cell cycle. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2224–2228. doi: 10.1073/pnas.69.8.2224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pederson T., Davis N. G. Messenger RNA processing and nuclear structure: isolation of nuclear ribonucleoprotein particles containing beta-globin messenger RNA precursors. J Cell Biol. 1980 Oct;87(1):47–54. doi: 10.1083/jcb.87.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pederson T. Gene activation in eukaryotes: are nuclear acidic proteins the cause or the effect? Proc Natl Acad Sci U S A. 1974 Mar;71(3):617–621. doi: 10.1073/pnas.71.3.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Pederson T. Messenger RNA biosynthesis and nuclear structure. Am Sci. 1981 Jan-Feb;69(1):76–84. [PubMed] [Google Scholar]
  42. 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]
  43. Pederson T., Robbins E. A method for improving synchrony in the G2 phase of the cell cycle. J Cell Biol. 1971 Jun;49(3):942–945. doi: 10.1083/jcb.49.3.942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Perry R. P., Kelley D. E. Buoyant densities of cytoplasmic ribonucleoprotein particles of mammalian cells: distinctive character of ribosome subunits and the rapidly labeled components. J Mol Biol. 1966 Apr;16(2):255–268. doi: 10.1016/s0022-2836(66)80171-7. [DOI] [PubMed] [Google Scholar]
  45. Salditt-Georgieff M., Harpold M., Sawicki S., Nevins J., Darnell J. E., Jr Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis. J Cell Biol. 1980 Sep;86(3):844–848. doi: 10.1083/jcb.86.3.844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Sen A., Todaro G. J. The genome-associated, specific RNA binding proteins of avian and mammalian type C viruses. Cell. 1977 Jan;10(1):91–99. doi: 10.1016/0092-8674(77)90143-x. [DOI] [PubMed] [Google Scholar]
  49. Smith K. C., Meun D. H. Kinetics of the photochemical addition of [35S] cysteine to polynucleotides and nucleic acids. Biochemistry. 1968 Mar;7(3):1033–1037. doi: 10.1021/bi00843a023. [DOI] [PubMed] [Google Scholar]
  50. Stevens B. J., Swift H. RNA transport from nucleus to cytoplasm in Chironomus salivary glands. J Cell Biol. 1966 Oct;31(1):55–77. doi: 10.1083/jcb.31.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]

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