Abstract
The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody- precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA- protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.
Full Text
The Full Text of this article is available as a PDF (960.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Avvedimento V. E., Vogeli G., Yamada Y., Maizel J. V., Jr, Pastan I., de Crombrugghe B. Correlation between splicing sites within an intron and their sequence complementarity with U1 RNA. Cell. 1980 Oct;21(3):689–696. doi: 10.1016/0092-8674(80)90432-8. [DOI] [PubMed] [Google Scholar]
- Branlant C., Krol A., Ebel J. P., Lazar E., Gallinaro H., Jacob M., Sri-Widada J., Jeanteur P. Nucleotide sequences of nuclear U1A RNAs from chicken, rat and man. Nucleic Acids Res. 1980 Sep 25;8(18):4143–4154. doi: 10.1093/nar/8.18.4143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Calvet J. P., Pederson T. Base-pairing interactions between small nuclear RNAs and nuclear RNA precursors as revealed by psoralen cross-linking in vivo. Cell. 1981 Nov;26(3 Pt 1):363–370. doi: 10.1016/0092-8674(81)90205-1. [DOI] [PubMed] [Google Scholar]
- De Robertis E. M., Lienhard S., Parisot R. F. Intracellular transport of microinjected 5S and small nuclear RNAs. Nature. 1982 Feb 18;295(5850):572–577. doi: 10.1038/295572a0. [DOI] [PubMed] [Google Scholar]
- Douvas A. S., Stumph W. E., Reyes P., Tan E. M. Isolation and characterization of nuclear ribonucleoprotein complexes using human anti-nuclear ribonucleoprotein antibodies. J Biol Chem. 1979 May 10;254(9):3608–3616. [PubMed] [Google Scholar]
- Kole R., Altman S. Reconstitution of RNase P activity from inactive RNA and protein. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3795–3799. doi: 10.1073/pnas.76.8.3795. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lazar E., Jacob M., Krol A., Branlant C. Accessibility of U1 RNA to base pairing with a single-stranded DNA fragment mimicking the intron extremities at the splice junction. Nucleic Acids Res. 1982 Feb 25;10(4):1193–1201. doi: 10.1093/nar/10.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lerner M. R., Boyle J. A., Mount S. M., Wolin S. L., Steitz J. A. Are snRNPs involved in splicing? Nature. 1980 Jan 10;283(5743):220–224. doi: 10.1038/283220a0. [DOI] [PubMed] [Google Scholar]
- Lerner M. R., Steitz J. A. Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5495–5499. doi: 10.1073/pnas.76.11.5495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nierhaus K. H., Dohme F. Total reconstitution of functionally active 50S ribosomal subunits from Escherichia coli. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4713–4717. doi: 10.1073/pnas.71.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
- Raj N. B., Ro-Choi T. S., Busch H. Nuclear ribonucleoprotein complexes containing U1 and U2 RNA. Biochemistry. 1975 Oct 7;14(20):4380–4385. doi: 10.1021/bi00691a006. [DOI] [PubMed] [Google Scholar]
- Rao M. S., Blackstone M., Busch H. Effects of U1 nuclear RNA on translation of messenger RNA. Biochemistry. 1977 Jun 14;16(12):2756–2762. doi: 10.1021/bi00631a026. [DOI] [PubMed] [Google Scholar]
- Rogers J., Wall R. A mechanism for RNA splicing. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1877–1879. doi: 10.1073/pnas.77.4.1877. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wieben E. D., Pederson T. Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock. Mol Cell Biol. 1982 Aug;2(8):914–920. doi: 10.1128/mcb.2.8.914. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wieben E. D., Pederson T. Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock. Mol Cell Biol. 1982 Aug;2(8):914–920. doi: 10.1128/mcb.2.8.914. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wieben E. D. Regulation of the synthesis of lactate dehydrogenase-X during spermatogenesis in the mouse. J Cell Biol. 1981 Mar;88(3):492–498. doi: 10.1083/jcb.88.3.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yang V. W., Lerner M. R., Steitz J. A., Flint S. J. A small nuclear ribonucleoprotein is required for splicing of adenoviral early RNA sequences. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1371–1375. doi: 10.1073/pnas.78.3.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zieve G., Penman S. Small RNA species of the HeLa cell: metabolism and subcellular localization. Cell. 1976 May;8(1):19–31. doi: 10.1016/0092-8674(76)90181-1. [DOI] [PubMed] [Google Scholar]