Skip to main content
PMC home page
RNA logoLink to RNA
. 1999 Nov;5(11):1509–1516. doi: 10.1017/s1355838299991574

StreptoTag: a novel method for the isolation of RNA-binding proteins.

M Bachler 1, R Schroeder 1, U von Ahsen 1
PMCID: PMC1369873  PMID: 10580480

Abstract

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA-protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA-protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, "StreptoTag," should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins.

Full Text

The Full Text of this article is available as a PDF (660.2 KB).

Selected References

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

  1. Bashirullah A., Cooperstock R. L., Lipshitz H. D. RNA localization in development. Annu Rev Biochem. 1998;67:335–394. doi: 10.1146/annurev.biochem.67.1.335. [DOI] [PubMed] [Google Scholar]
  2. Cusack S. RNA-protein complexes. Curr Opin Struct Biol. 1999 Feb;9(1):66–73. doi: 10.1016/s0959-440x(99)80009-8. [DOI] [PubMed] [Google Scholar]
  3. Gold L., Singer B., He Y. Y., Brody E. SELEX and the evolution of genomes. Curr Opin Genet Dev. 1997 Dec;7(6):848–851. doi: 10.1016/s0959-437x(97)80050-0. [DOI] [PubMed] [Google Scholar]
  4. Hall K. B., Stump W. T. Interaction of N-terminal domain of U1A protein with an RNA stem/loop. Nucleic Acids Res. 1992 Aug 25;20(16):4283–4290. doi: 10.1093/nar/20.16.4283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Holeman L. A., Robinson S. L., Szostak J. W., Wilson C. Isolation and characterization of fluorophore-binding RNA aptamers. Fold Des. 1998;3(6):423–431. doi: 10.1016/S1359-0278(98)00059-5. [DOI] [PubMed] [Google Scholar]
  6. Iyer V., Struhl K. Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5208–5212. doi: 10.1073/pnas.93.11.5208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Krecic A. M., Swanson M. S. hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol. 1999 Jun;11(3):363–371. doi: 10.1016/S0955-0674(99)80051-9. [DOI] [PubMed] [Google Scholar]
  8. Küster B., Mann M. Identifying proteins and post-translational modifications by mass spectrometry. Curr Opin Struct Biol. 1998 Jun;8(3):393–400. doi: 10.1016/s0959-440x(98)80075-4. [DOI] [PubMed] [Google Scholar]
  9. Lindqvist Y., Schneider G. Protein-biotin interactions. Curr Opin Struct Biol. 1996 Dec;6(6):798–803. doi: 10.1016/s0959-440x(96)80010-8. [DOI] [PubMed] [Google Scholar]
  10. Lowary P. T., Uhlenbeck O. C. An RNA mutation that increases the affinity of an RNA-protein interaction. Nucleic Acids Res. 1987 Dec 23;15(24):10483–10493. doi: 10.1093/nar/15.24.10483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Minvielle-Sebastia L., Keller W. mRNA polyadenylation and its coupling to other RNA processing reactions and to transcription. Curr Opin Cell Biol. 1999 Jun;11(3):352–357. doi: 10.1016/S0955-0674(99)80049-0. [DOI] [PubMed] [Google Scholar]
  12. Mumberg D., Müller R., Funk M. Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res. 1994 Dec 25;22(25):5767–5768. doi: 10.1093/nar/22.25.5767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Neupert B., Thompson N. A., Meyer C., Kühn L. C. A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta. Nucleic Acids Res. 1990 Jan 11;18(1):51–55. doi: 10.1093/nar/18.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rouault T. A., Hentze M. W., Haile D. J., Harford J. B., Klausner R. D. The iron-responsive element binding protein: a method for the affinity purification of a regulatory RNA-binding protein. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5768–5772. doi: 10.1073/pnas.86.15.5768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Siomi H., Dreyfuss G. RNA-binding proteins as regulators of gene expression. Curr Opin Genet Dev. 1997 Jun;7(3):345–353. doi: 10.1016/s0959-437x(97)80148-7. [DOI] [PubMed] [Google Scholar]
  16. Stripecke R., Oliveira C. C., McCarthy J. E., Hentze M. W. Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells. Mol Cell Biol. 1994 Sep;14(9):5898–5909. doi: 10.1128/mcb.14.9.5898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wallace S. T., Schroeder R. In vitro selection and characterization of streptomycin-binding RNAs: recognition discrimination between antibiotics. RNA. 1998 Jan;4(1):112–123. [PMC free article] [PubMed] [Google Scholar]
  18. Werstuck G., Green M. R. Controlling gene expression in living cells through small molecule-RNA interactions. Science. 1998 Oct 9;282(5387):296–298. doi: 10.1126/science.282.5387.296. [DOI] [PubMed] [Google Scholar]
  19. Witherell G. W., Gott J. M., Uhlenbeck O. C. Specific interaction between RNA phage coat proteins and RNA. Prog Nucleic Acid Res Mol Biol. 1991;40:185–220. doi: 10.1016/s0079-6603(08)60842-9. [DOI] [PubMed] [Google Scholar]
  20. von Ahsen U., Noller H. F. Identification of bases in 16S rRNA essential for tRNA binding at the 30S ribosomal P site. Science. 1995 Jan 13;267(5195):234–237. doi: 10.1126/science.7528943. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES