Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Oct 1;25(19):3816–3822. doi: 10.1093/nar/25.19.3816

Detection and isolation of RNA-binding proteins by RNA-ligand screening of a cDNA expression library.

R Sägesser 1, E Martinez 1, M Tsagris 1, M Tabler 1
PMCID: PMC146985  PMID: 9380503

Abstract

A screening assay for the detection of RNA-binding proteins was developed. It allows the rapid isolation of cDNA clones coding for proteins with sequence-specific binding affinity to a target RNA. For developing the screening protocol, constituents of the human U1 snRNP were utilized as model system. The RNA partner consisted of the U1-RNA stem-loop II and the corresponding protein consisted of the 102 amino acid N-terminal recognition motif of the U1A protein, which was fused to beta-galactosidase and expressed by the recombinant lambda phage LU1A. Following binding of the fusion protein to nitrocellulose membranes, hybridization with a 32P-labeled U1-RNA ligand was carried out to detect specific RNA-protein interaction. Parameters influencing the specificity and the detection limit of binding were systematically investigated with the aid of the model system. Processing the nitrocellulose membranes in the presence of transition metals greatly increased the signal:background ratio. A simple screening protocol involving a single-buffer system was developed. Specific RNA-protein interaction could be detected in the presence of a large excess of recombinant phages from a cDNA library. Only moderate binding affinities (Kd = 10(-8) M) were required. The suitability of the RNA-ligand screening protocol was demonstrated by the identification of new viroid-RNA binding proteins from tomato.

Full Text

The Full Text of this article is available as a PDF (154.0 KB).

Selected References

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

  1. Harada K., Martin S. S., Frankel A. D. Selection of RNA-binding peptides in vivo. Nature. 1996 Mar 14;380(6570):175–179. doi: 10.1038/380175a0. [DOI] [PubMed] [Google Scholar]
  2. Jessen T. H., Oubridge C., Teo C. H., Pritchard C., Nagai K. Identification of molecular contacts between the U1 A small nuclear ribonucleoprotein and U1 RNA. EMBO J. 1991 Nov;10(11):3447–3456. doi: 10.1002/j.1460-2075.1991.tb04909.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Laird-Offringa I. A., Belasco J. G. Analysis of RNA-binding proteins by in vitro genetic selection: identification of an amino acid residue important for locking U1A onto its RNA target. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11859–11863. doi: 10.1073/pnas.92.25.11859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Laird-Offringa I. A., Belasco J. G. In vitro genetic analysis of RNA-binding proteins using phage display libraries. Methods Enzymol. 1996;267:149–168. doi: 10.1016/s0076-6879(96)67011-6. [DOI] [PubMed] [Google Scholar]
  5. MacWilliams M. P., Celander D. W., Gardner J. F. Direct genetic selection for a specific RNA-protein interaction. Nucleic Acids Res. 1993 Dec 11;21(24):5754–5760. doi: 10.1093/nar/21.24.5754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Prombona A., Tabler M., Providaki M., Tsagris M. Structure and expression of LeMA-1, a tomato protein belonging to the SEC18-PAS1-CDC48-TBP-1 protein family of putative Mg(2+)-dependent ATPases. Plant Mol Biol. 1995 Mar;27(6):1109–1118. doi: 10.1007/BF00020884. [DOI] [PubMed] [Google Scholar]
  7. Putz U., Skehel P., Kuhl D. A tri-hybrid system for the analysis and detection of RNA--protein interactions. Nucleic Acids Res. 1996 Dec 1;24(23):4838–4840. doi: 10.1093/nar/24.23.4838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Qian Z., Wilusz J. Cloning of a cDNA encoding an RNA binding protein by screening expression libraries using a northwestern strategy. Anal Biochem. 1993 Aug 1;212(2):547–554. doi: 10.1006/abio.1993.1367. [DOI] [PubMed] [Google Scholar]
  9. Scherly D., Boelens W., van Venrooij W. J., Dathan N. A., Hamm J., Mattaj I. W. Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA. EMBO J. 1989 Dec 20;8(13):4163–4170. doi: 10.1002/j.1460-2075.1989.tb08601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. SenGupta D. J., Zhang B., Kraemer B., Pochart P., Fields S., Wickens M. A three-hybrid system to detect RNA-protein interactions in vivo. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8496–8501. doi: 10.1073/pnas.93.16.8496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Singh H., LeBowitz J. H., Baldwin A. S., Jr, Sharp P. A. Molecular cloning of an enhancer binding protein: isolation by screening of an expression library with a recognition site DNA. Cell. 1988 Feb 12;52(3):415–423. doi: 10.1016/s0092-8674(88)80034-5. [DOI] [PubMed] [Google Scholar]
  12. Symons R. H. The intriguing viroids and virusoids: what is their information content and how did they evolve? Mol Plant Microbe Interact. 1991 Mar-Apr;4(2):111–121. doi: 10.1094/mpmi-4-111. [DOI] [PubMed] [Google Scholar]
  13. Tabler M., Sänger H. L. Infectivity studies on different potato spindle tuber viroid (PSTV) RNAs synthesized in vitro with the SP6 transcription system. EMBO J. 1985 Sep;4(9):2191–2199. doi: 10.1002/j.1460-2075.1985.tb03914.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Tabler M., Tsagris M. Catalytic antisense RNAs produced by incorporating ribozyme cassettes into cDNA. Gene. 1991 Dec 15;108(2):175–183. doi: 10.1016/0378-1119(91)90432-b. [DOI] [PubMed] [Google Scholar]
  15. Tsagris M., Tabler M., Sänger H. L. Ribonuclease T1 generates circular RNA molecules from viroid-specific RNA transcripts by cleavage and intramolecular ligation. Nucleic Acids Res. 1991 Apr 11;19(7):1605–1612. doi: 10.1093/nar/19.7.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Vinson C. R., LaMarco K. L., Johnson P. F., Landschulz W. H., McKnight S. L. In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage. Genes Dev. 1988 Jul;2(7):801–806. doi: 10.1101/gad.2.7.801. [DOI] [PubMed] [Google Scholar]
  17. Werner R., Mühlbach H. P., Guitton M. C. Isolation of viroid-RNA-binding proteins from an expression library with nonradioactive-labeled RNA probes. Biotechniques. 1995 Aug;19(2):218–222. [PubMed] [Google Scholar]
  18. Zaidi S. H., Malter J. S. Nucleolin and heterogeneous nuclear ribonucleoprotein C proteins specifically interact with the 3'-untranslated region of amyloid protein precursor mRNA. J Biol Chem. 1995 Jul 21;270(29):17292–17298. doi: 10.1074/jbc.270.29.17292. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES