Abstract
The majority of the protein mass of HeLa 40S heterogeneous nuclear ribonucleoprotein monoparticles is composed of multiple copies of six proteins that resolve in SDS gels as three groups of doublet bands (A1, A2; B1, B2; and C1, C2) (Beyer, A. L., M. E. Christensen, B. W. Walker, and W. M. LeStourgeon. 1977. Cell. 11: 127-138). We report here that when 40S monoparticles are exposed briefly to ribonuclease, proteins A1, C1, and C2 are solubilized coincidentally with the loss of most premessenger RNA sequences. The remaining proteins exist as tetramers of (A2)3(B1) or pentamers of (A2)3(B1)(B2). The tetramers may reassociate in highly specific ways to form either of two different structures. In 0.1 M salt approximately 12 tetramers (derived from three or four monoparticles) reassemble to form highly regular structures, which may possess dodecahedral symmetry. These structures sediment at 43S, are 20-22 nm in width, and have a mass near 2.3 million. These structures possess 450-500 bases of slowly labeled RNA, which migrates in gels as fragments 200-220 bases in length. In 9 mM salt the tetramers reassociate to form 2.0 M salt-insoluble helical filaments of indeterminant length with a pitch near 60 nm and diameter near 18 nm. If 40S monoparticles are treated briefly with nuclease-free proteases, the same proteins solubilized by nuclease (A1, C1, and C2) are preferentially cleaved. This protein cleavage is associated with the dissociation of most of the heterogeneous nuclear RNA. Proteins A2 and B1 again reassemble to form uniform, globular particles, but these sediment slightly slower than intact monoparticles. These findings indicate that proteins A1, C1, and C2 and most of the premessenger sequences occupy a peripheral position in intact monoparticles and that their homotypic and heterotypic associations are dependent on protein- RNA interactions. Protein cross-linking studies demonstrate that trimers of A1, A2, and C1 exist as the most easily stabilized homotypic association in 40S particles. This supports the 3:1 ratio (via densitometry) of the A and C proteins to the B proteins and indicates that 40S monoparticles are composed of three or four repeating units, each containing 3(A1),3(A2),1(B1),1(B2),3(C1), and 1(C2).
Full Text
The Full Text of this article is available as a PDF (1.6 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- 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]
- 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]
- 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]
- Dubochet J., Kellenberger E. Selective adsorption of particles to the supporting film and its consequences on particle counts in electron microscopy. Microsc Acta. 1972 Jul;72(2):119–130. [PubMed] [Google Scholar]
- Economidis I. V., Pederson T. Structure of nuclear ribonucleoprotein: heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1599–1602. doi: 10.1073/pnas.80.6.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldenberg D. P., Berget P. B., King J. Maturation of the tail spike endorhamnosidase of Salmonella phage P22. J Biol Chem. 1982 Jul 10;257(13):7864–7871. [PubMed] [Google Scholar]
- 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]
- 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]
- Kloetzel P. M., Johnson R. M., Sommerville J. Interaction of the hnRNA of amphibian oocytes with fibril-forming proteins. Eur J Biochem. 1982 Oct;127(2):301–308. doi: 10.1111/j.1432-1033.1982.tb06870.x. [DOI] [PubMed] [Google Scholar]
- Knowler J. T. An assessment of the evidence for the role of ribonucleoprotein particles in the maturation of eukaryote mRNA. Int Rev Cytol. 1983;84:103–153. doi: 10.1016/s0074-7696(08)61016-5. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- LeStourgeon W. M., Beyer A. L., Christensen M. E., Walker B. W., Poupore S. M., Daniels L. P. The packaging proteins of core hnRNP particles and the maintenance of proliferative cell states. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):885–898. doi: 10.1101/sqb.1978.042.01.090. [DOI] [PubMed] [Google Scholar]
- LeStourgeon W. M., Beyer A. L. The rapid isolation, high-resolution electrophoretic characterization, and purification of nuclear proteins. Methods Cell Biol. 1977;16:387–406. [PubMed] [Google Scholar]
- Loeb J. E., Ritz E., Creuzet C., Jami J. Comparison of chromosomal proteins of mouse primitive teratocarcinoma, liver and L cells. Exp Cell Res. 1976 Dec;103(2):450–453. doi: 10.1016/0014-4827(76)90287-1. [DOI] [PubMed] [Google Scholar]
- Martin T., Billings P., Levey A., Ozarslan S., Quinlan T., Swift H., Urbas L. Some properties of RNA:protein complexes from the nucleus of eukaryotic cells. Cold Spring Harb Symp Quant Biol. 1974;38:921–932. doi: 10.1101/sqb.1974.038.01.094. [DOI] [PubMed] [Google Scholar]
- 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]
- Pederson T. Nuclear RNA-protein interactions and messenger RNA processing. J Cell Biol. 1983 Nov;97(5 Pt 1):1321–1326. doi: 10.1083/jcb.97.5.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Peters K. E., Commings D. E. Two-dimensinal gel electrophoresis of rat liver nuclear washes, nuclear matrix, and hnRNA proteins. J Cell Biol. 1980 Jul;86(1):135–155. doi: 10.1083/jcb.86.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Spirin A. S. The second Sir Hans Krebs Lecture. Informosomes. Eur J Biochem. 1969 Aug;10(1):20–35. doi: 10.1111/j.1432-1033.1969.tb00651.x. [DOI] [PubMed] [Google Scholar]
- Steitz J. A., Kamen R. Arrangement of 30S heterogeneous nuclear ribonucleoprotein on polyoma virus late nuclear transcripts. Mol Cell Biol. 1981 Jan;1(1):21–34. doi: 10.1128/mcb.1.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. 1977 Apr 15;74(3):589–602. doi: 10.1111/j.1432-1033.1977.tb11428.x. [DOI] [PubMed] [Google Scholar]
- Stevenin J., Jacob M. Structure of premRNP. Models and pitfalls. Mol Biol Rep. 1979 May 31;5(1-2):29–35. doi: 10.1007/BF00777485. [DOI] [PubMed] [Google Scholar]
- Stévenin J., Gattoni R., Divilliers G., Jacob M. Rearrangements in the course of ribonuclease hydrolysis of pre-messenger ribonucleoproteins. A warning. Eur J Biochem. 1979 Apr;95(3):593–606. doi: 10.1111/j.1432-1033.1979.tb13000.x. [DOI] [PubMed] [Google Scholar]
- Tsanev R. G., Djondjurov L. P. Ultrastructure of free ribonucleoprotein complexes in spread mammalian nuclei. J Cell Biol. 1982 Sep;94(3):662–666. doi: 10.1083/jcb.94.3.662. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walker B. W., Lothstein L., Baker C. L., LeStourgeon W. M. The release of 40S hnRNP particles by brief digestion of HeLa nuclei with micrococcal nuclease. Nucleic Acids Res. 1980 Aug 25;8(16):3639–3657. doi: 10.1093/nar/8.16.3639. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang K., Richards F. M. An approach to nearest neighbor analysis of membrane proteins. Application to the human erythrocyte membrane of a method employing cleavable cross-linkages. J Biol Chem. 1974 Dec 25;249(24):8005–8018. [PubMed] [Google Scholar]
- Wilk H. E., Angeli G., Schäfer K. P. In vitro reconstitution of 35S ribonucleoprotein complexes. Biochemistry. 1983 Sep 13;22(19):4592–4600. doi: 10.1021/bi00288a038. [DOI] [PubMed] [Google Scholar]
- 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]