Summary
The nature of the association of U1 RNA with rapidly sedimenting RNP structures in rat hepatoma nuclei was investigated. The effects of salt and proteinase K treatment on the stability of this ‘bound’ form of U1 RNA were studied using sucrose density gradient analyses. Quantitation of the amount of U1 RNA remaining associated with large structures after treatment was used to assess the relative contribution of protein-protein(and protein-RNA) versus RNA-RNA interactions. Forty-eight percent of the total nuclear U1 RNA released by sonication was found in a ‘bound’ form when the sonicate was centrifuged through gradients containing 50 mM NaCl. Fifty percent of this ‘bound’ U1 RNA remained associated with rapidly sedimenting RNPs when the NaCl concentration was raised to 500 mM. To assess the contribution of protein independent interactions, large RNPs were completely deproteinized and their RNA moieties were then recentrifuged on gradients. By this analysis, 27% of the U1 RNA originally ‘bound’ to hnRNPs was associated with rapidly sedimenting (>30 S) RNA (at 50 mM NaCl) suggesting their association by RNA-RNA hydrogen bonds. When the concentration of NaCl was 500 mM, 31% of the U1 RNA was associated with large RNA. Hence, approximately 30% of the U1 RNA molecules originally ‘bound’ (or about 15% of the total nuclear U1 RNA) were found to be associated by RNA-RNA hydrogen bonds while the remainder of the binding of U1 RNP to hnRNP was by protein-protein and/or protein-RNA interactions.
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References
Knowler JT: An assessment of the evidence for the role of ribonucleoprotein particles in the maturation of eukaryote mRNA. Int Rev Cytol 84:103–153, 1983.
Stevenin J, Gallinaro-Matringe H, Gattoni R, Jacob M: Complexity of the structure of particles containing heterogeneous nuclear RNA as demonstrated by ribonuclease treatment. Eur J Biochem 74:589–602, 1977.
Heinrich PC, Northemann W: The structure of ribonucleo-protein particles from rat liver nuclei. Mol Biol Rep 7:15–24, 1981.
Samarina OP, Krichevskaya AA: Nuclear 30S RNP particles. In: H Busch (ed), The Cell Nucleus, Vol IX. Academic Press, New York, 1981, pp 1–48.
Zieve G, Penman S: Subnuclear particles containing small nuclear RNA and heterogeneous nuclear RNA. J Mol Biol 145:501–523, 1981.
Fuchs JP, Jacob M: Fractionation of constituents of ribonucleoproteins containing heterogeneous nuclear ribonucleic acid. Biochemistry 18:4202–4208, 1979.
Economidis IV, Pederson T: Structure of nuclear ribonucleoprotein: Heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo. Proc Natl Acad Sci USA 80:1599–1602, 1983.
Jacob M, Devilliers G, Fuchs JP, Gallinaro H, Gattoni R, Judes C, Stevenin J: Isolation and structure of the ribonucleoprotein fibrils containing heterogeneous nuclear RNA. In: H Busch (ed), The Cell Nucleus, Vol VIII. Academic Press, New York, 1981, pp 193–246.
Pederson T: Nuclear RNA-protein interactions and messenger RNA processing. J Cell Biol 97:1321–1326, 1983.
Augenlicht LH: Staphylococcal nuclease digestion of relatively stable RNA in the chromatin and nuclear ribonucleoprotein fractions of human carcinoma cells. J Biol Chem 253:3035–3041, 1968.
Reddy R, Busch H: U snRNAs of nuclear snRNPs. In: H Busch (ed), The Cell Nucleus, Vol VIII. Academic Press, New York, 1981, pp 261–306.
Busch H, Reddy R, Rothblum L, Choi Y: SnRNAs, snRNPs, and RNA processing. Ann Rev Biochem 51:617–654, 1982.
Liautard JP, Sri-Widada J, Brunel C, Jeanteur P: Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. J Mol Biol 162:623–643, 1982.
Flytzanis C, Alonso A, Louis C, Krieg L, Sekeris C: Association of small nuclear RNA with hnRNA isolated from nuclear RNP complexes carrying hnRNA. FEBS Lett 96:201–206, 1978.
Calvet JP, Pederson T: Base-pairing interactions between small nuclear RNAs and nuclear RNA precursors as revealed by psoralen crosslinking. Cell 26:363–370, 1981.
Calvet JP, Meyer LM, Pederson T: Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA. Science 217:456–458, 1982.
Gallinaro H, Jacob M: The status of small nuclear RNA in the ribonucleoprotein fibrils containing heterogeneous nuclear RNA. Biochim Biophys Acta 652:109–120, 1981.
Northemann W, Klump H, Heinrich PC: The effect of urea on the structure of nuclear ribonucleoprotein particles from rat liver. Eur J Biochem 99:447–456, 1979.
Rogers J, Wall R: A mechanism for RNA splicing. Proc Natl Acad Sci USA 77:1877–1879, 1980.
Yang VW, Lerner M, Steitz J, Flint J: A small nuclear ribonucleoprotein is required for splicing of adenoviral early RNA sequences. Proc Natl Acad Sci USA 78:1371–1375, 1981.
Mount S, Petterson I, Hinterberger M, Karmas A, Steitz J: The U1 small nuclear RNA-protein complex selectively binds a 5′ splice site in vitro. Cell 33:509–518, 1983.
Wu BC, Spohn WH, Busch H: Two-dimensional gel electrophoresis of cytosol phosphoproteins of Novikoff hepatoma and regenerating liver. Cancer Res 39:116–122, 1979.
Yeoman LC, Jordan JJ, Busch RK, Taylor CW, Savage H, Busch H: A fetal protein in chromatin of Novikoff hepatoma and Walker 256 carcinosarcoma tumors that is absent from normal and regenerating rat liver. Proc Natl Acad Sci USA 73:3258–3262, 1976.
Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254, 1976.
McEwen CR: Tables for estimating sedimentation through linear concentration gradients of sucrose solution. Anal Biochem 20:114–149, 1967.
McMaster GK, Carmichael GG: Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci USA 74:4835–4838, 1977.
Roop DR, Kristo P, Stumpf WE, Tsai MJ, O'Malley BW: Structure and expression of a chicken gene coding for U1 RNA. Cell 23:671–680, 1981.
Gallinaro H, Lazar E, Jacob M, Krol A, Branlant C: Small RNAs in hnRNP fibrils and their possible function in splicing. Mol Biol Rep 7:31–39, 1981.
Northemann W, Gross V, Scheurlen M, Heinrich PC: Studies on the preparation and properties of ribonucleoprotein particles from rat liver nuclei. Biochim Biophys Acta 519:406–417, 1978.
Northemann W, Seifert H, Heinrich PC: The effect of sodium chloride on the structure of ribonucleoprotein particles from rat liver nuclei. Hoppe-Seylers Z Physiol Chem 360:877–888, 1979.
Brunel C, Sri Widada J, Lelay M-N, Jeanteur P, Liautard J-P: Purification and characterization of a simple ribonucleoprotein particle containing small nucleoplasmic RNAs (snRNP) as a subset of RNP containing heterogenous nuclear RNA (hnRNP) from HeLa cells. Nucl Acds Res 9:815–830, 1981.
Sri Widada J, Assens C, Liautard J-P, Jeanteur P, Brunel C: Isolation of a pure U1 snRNP from HeLa cells. Biochem Biophys Res Commun 104:457–462, 1982.
Lerner M, Boyle J, Mount S, Wolin S, Steitz J: Are snRNPs involved in splicing? Nature 283:220–224, 1980.
Prusse A, Louis C, Alonso A, Sekeris C: Isolation and characterization of hnRNA-snRNA-protein complexes from Morris hepatoma cells. Eur J Biochem 128:169–178, 1982.
Baltimore D, Huang AS: Interaction of HeLa cell proteins with RNA. J Mol Biol 47:263–273, 1970.
Pogo OA: Heterogeneous nuclear RNA protein complexes and nuclear matrix. In: H Busch (ed), The Cell Nucleus, Vol XIII. Academic Press, New York, 1981, pp 331–367.
Epstein P, Reddy R, Busch H: Site-specific cleavage by T1 RNase of U-1 RNA in U-1 ribonucleoprotein particles. Proc Natl Acad Sci USA 78:1562–1566, 1981.
Padgett RA, Mount SM, Steitz JA, Sharp PA: Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein. Cell 35:101–107, 1983.
Mount SM, Steitz JA: Sequence of U1 RNA from Drosophila melanogaster: implications for U1 secondary structure and possible involvement in splicing. Nucl Acids Res 9:6351–6368, 1981.
Ohlsson RI, vanEekelan C, Philipson L: Non-random localization of ribonucleoprotein structures within an adenovirus mRNA precursor. Nucl Acids Res 10:3053–3068, 1982.
Shenk TE, Carbon J, Berg PL Construction and analysis of viable deletion mutants of simian virus 40. J Virol 18:664–671, 1976.
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Harmon, F.R., Subrahmanyam, C.S. & Busch, H. Interactions of U1 RNP with heterogeneous nuclear RNA in rat Novikoff hepatoma nuclei. Mol Cell Biochem 65, 45–55 (1984). https://doi.org/10.1007/BF00226018
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DOI: https://doi.org/10.1007/BF00226018