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. 2002 Sep;8(9):1160–1173. doi: 10.1017/s135583820202808x

Targeting a KH-domain protein with RNA decoys.

Aleksandr V Makeyev 1, Dawn L Eastmond 1, Stephen A Liebhaber 1
PMCID: PMC1370330  PMID: 12358435

Abstract

RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to alphaCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for alphaCP decoy action. Because alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of alphaCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins.

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Selected References

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  1. Agrawal S. Antisense oligonucleotides: towards clinical trials. Trends Biotechnol. 1996 Oct;14(10):376–387. doi: 10.1016/0167-7799(96)10053-6. [DOI] [PubMed] [Google Scholar]
  2. Andino R., Böddeker N., Silvera D., Gamarnik A. V. Intracellular determinants of picornavirus replication. Trends Microbiol. 1999 Feb;7(2):76–82. doi: 10.1016/s0966-842x(98)01446-2. [DOI] [PubMed] [Google Scholar]
  3. Barcellini-Couget S., Fenard D., Bertrand E., Singer R. H., Lefebvre J. C., Doglio A. 3'-End modification of the adenoviral VA1 gene affects its expression in human cells: consequences for the design of chimeric VA1 RNA ribozymes. Antisense Nucleic Acid Drug Dev. 1998 Oct;8(5):379–390. doi: 10.1089/oli.1.1998.8.379. [DOI] [PubMed] [Google Scholar]
  4. Bertrand E., Castanotto D., Zhou C., Carbonnelle C., Lee N. S., Good P., Chatterjee S., Grange T., Pictet R., Kohn D. The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization. RNA. 1997 Jan;3(1):75–88. [PMC free article] [PubMed] [Google Scholar]
  5. Bertrand E., Pictet R., Grange T. Can hammerhead ribozymes be efficient tools to inactivate gene function? Nucleic Acids Res. 1994 Feb 11;22(3):293–300. doi: 10.1093/nar/22.3.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blyn L. B., Towner J. S., Semler B. L., Ehrenfeld E. Requirement of poly(rC) binding protein 2 for translation of poliovirus RNA. J Virol. 1997 Aug;71(8):6243–6246. doi: 10.1128/jvi.71.8.6243-6246.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bohjanen P. R., Liu Y., Garcia-Blanco M. A. TAR RNA decoys inhibit tat-activated HIV-1 transcription after preinitiation complex formation. Nucleic Acids Res. 1997 Nov 15;25(22):4481–4486. doi: 10.1093/nar/25.22.4481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng X., DeLong R. K., Wickstrom E., Kligshteyn M., Demirdji S. H., Caruthers M. H., Juliano R. L. Interactions between single-stranded DNA binding protein and oligonucleotide analogs with different backbone chemistries. J Mol Recognit. 1997 Mar-Apr;10(2):101–107. doi: 10.1002/(SICI)1099-1352(199703/04)10:2<101::AID-JMR344>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
  9. Chkheidze A. N., Lyakhov D. L., Makeyev A. V., Morales J., Kong J., Liebhaber S. A. Assembly of the alpha-globin mRNA stability complex reflects binary interaction between the pyrimidine-rich 3' untranslated region determinant and poly(C) binding protein alphaCP. Mol Cell Biol. 1999 Jul;19(7):4572–4581. doi: 10.1128/mcb.19.7.4572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Collier B., Goobar-Larsson L., Sokolowski M., Schwartz S. Translational inhibition in vitro of human papillomavirus type 16 L2 mRNA mediated through interaction with heterogenous ribonucleoprotein K and poly(rC)-binding proteins 1 and 2. J Biol Chem. 1998 Aug 28;273(35):22648–22656. doi: 10.1074/jbc.273.35.22648. [DOI] [PubMed] [Google Scholar]
  11. Conn G. L., Draper D. E. RNA structure. Curr Opin Struct Biol. 1998 Jun;8(3):278–285. doi: 10.1016/s0959-440x(98)80059-6. [DOI] [PubMed] [Google Scholar]
  12. Czyzyk-Krzeska M. F., Bendixen A. C. Identification of the poly(C) binding protein in the complex associated with the 3' untranslated region of erythropoietin messenger RNA. Blood. 1999 Mar 15;93(6):2111–2120. [PubMed] [Google Scholar]
  13. Dyer R. B., Herzog N. K. Isolation of intact nuclei for nuclear extract preparation from a fragile B-lymphocyte cell line. Biotechniques. 1995 Aug;19(2):192–195. [PubMed] [Google Scholar]
  14. Ellington A. D., Szostak J. W. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990 Aug 30;346(6287):818–822. doi: 10.1038/346818a0. [DOI] [PubMed] [Google Scholar]
  15. Feldman H. A. Mathematical theory of complex ligand-binding systems of equilibrium: some methods for parameter fitting. Anal Biochem. 1972 Aug;48(2):317–338. doi: 10.1016/0003-2697(72)90084-x. [DOI] [PubMed] [Google Scholar]
  16. Funke B., Zuleger B., Benavente R., Schuster T., Goller M., Stévenin J., Horak I. The mouse poly(C)-binding protein exists in multiple isoforms and interacts with several RNA-binding proteins. Nucleic Acids Res. 1996 Oct 1;24(19):3821–3828. doi: 10.1093/nar/24.19.3821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Galabru J., Katze M. G., Robert N., Hovanessian A. G. The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase. Eur J Biochem. 1989 Jan 2;178(3):581–589. doi: 10.1111/j.1432-1033.1989.tb14485.x. [DOI] [PubMed] [Google Scholar]
  18. Gamarnik A. V., Andino R. Interactions of viral protein 3CD and poly(rC) binding protein with the 5' untranslated region of the poliovirus genome. J Virol. 2000 Mar;74(5):2219–2226. doi: 10.1128/jvi.74.5.2219-2226.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gamarnik A. V., Andino R. Switch from translation to RNA replication in a positive-stranded RNA virus. Genes Dev. 1998 Aug 1;12(15):2293–2304. doi: 10.1101/gad.12.15.2293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gamarnik A. V., Andino R. Two functional complexes formed by KH domain containing proteins with the 5' noncoding region of poliovirus RNA. RNA. 1997 Aug;3(8):882–892. [PMC free article] [PubMed] [Google Scholar]
  21. Ghadge G. D., Swaminathan S., Katze M. G., Thimmapaya B. Binding of the adenovirus VAI RNA to the interferon-induced 68-kDa protein kinase correlates with function. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7140–7144. doi: 10.1073/pnas.88.16.7140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hazelrigg T. The destinies and destinations of RNAs. Cell. 1998 Nov 13;95(4):451–460. doi: 10.1016/s0092-8674(00)81613-x. [DOI] [PubMed] [Google Scholar]
  23. Holcik M., Liebhaber S. A. Four highly stable eukaryotic mRNAs assemble 3' untranslated region RNA-protein complexes sharing cis and trans components. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2410–2414. doi: 10.1073/pnas.94.6.2410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hélène C., Toulmé J. J. Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim Biophys Acta. 1990 Jun 21;1049(2):99–125. doi: 10.1016/0167-4781(90)90031-v. [DOI] [PubMed] [Google Scholar]
  25. Ilves H., Barske C., Junker U., Böhnlein E., Veres G. Retroviral vectors designed for targeted expression of RNA polymerase III-driven transcripts: a comparative study. Gene. 1996 Jun 1;171(2):203–208. doi: 10.1016/0378-1119(96)00075-3. [DOI] [PubMed] [Google Scholar]
  26. Ishizaki J., Nevins J. R., Sullenger B. A. Inhibition of cell proliferation by an RNA ligand that selectively blocks E2F function. Nat Med. 1996 Dec;2(12):1386–1389. doi: 10.1038/nm1296-1386. [DOI] [PubMed] [Google Scholar]
  27. James W. Nucleic acid and polypeptide aptamers: a powerful approach to ligand discovery. Curr Opin Pharmacol. 2001 Oct;1(5):540–546. doi: 10.1016/s1471-4892(01)00093-5. [DOI] [PubMed] [Google Scholar]
  28. Jones M. H., Guthrie C. Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site. EMBO J. 1990 Aug;9(8):2555–2561. doi: 10.1002/j.1460-2075.1990.tb07436.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Katze M. G., DeCorato D., Safer B., Galabru J., Hovanessian A. G. Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity. EMBO J. 1987 Mar;6(3):689–697. doi: 10.1002/j.1460-2075.1987.tb04809.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kiledjian M., Wang X., Liebhaber S. A. Identification of two KH domain proteins in the alpha-globin mRNP stability complex. EMBO J. 1995 Sep 1;14(17):4357–4364. doi: 10.1002/j.1460-2075.1995.tb00110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Konopka K., Lee N. S., Rossi J., Düzgüneş N. Rev-binding aptamer and CMV promoter act as decoys to inhibit HIV replication. Gene. 2000 Sep 19;255(2):235–244. doi: 10.1016/s0378-1119(00)00334-6. [DOI] [PubMed] [Google Scholar]
  32. Lebruska L. L., Maher L. J., 3rd Selection and characterization of an RNA decoy for transcription factor NF-kappa B. Biochemistry. 1999 Mar 9;38(10):3168–3174. doi: 10.1021/bi982515x. [DOI] [PubMed] [Google Scholar]
  33. Lewandoski M. Conditional control of gene expression in the mouse. Nat Rev Genet. 2001 Oct;2(10):743–755. doi: 10.1038/35093537. [DOI] [PubMed] [Google Scholar]
  34. Makeyev A. V., Liebhaber S. A. Identification of two novel mammalian genes establishes a subfamily of KH-domain RNA-binding proteins. Genomics. 2000 Aug 1;67(3):301–316. doi: 10.1006/geno.2000.6244. [DOI] [PubMed] [Google Scholar]
  35. Matunis M. J., Michael W. M., Dreyfuss G. Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein. Mol Cell Biol. 1992 Jan;12(1):164–171. doi: 10.1128/mcb.12.1.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Miller P. S. Oligonucleoside methylphosphonates as antisense reagents. Biotechnology (N Y) 1991 Apr;9(4):358–362. doi: 10.1038/nbt0491-358. [DOI] [PubMed] [Google Scholar]
  37. Montgomery R. A., Dietz H. C. Inhibition of fibrillin 1 expression using U1 snRNA as a vehicle for the presentation of antisense targeting sequence. Hum Mol Genet. 1997 Apr;6(4):519–525. doi: 10.1093/hmg/6.4.519. [DOI] [PubMed] [Google Scholar]
  38. Ostareck D. H., Ostareck-Lederer A., Wilm M., Thiele B. J., Mann M., Hentze M. W. mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3' end. Cell. 1997 May 16;89(4):597–606. doi: 10.1016/s0092-8674(00)80241-x. [DOI] [PubMed] [Google Scholar]
  39. Paulding W. R., Czyzyk-Krzeska M. F. Regulation of tyrosine hydroxylase mRNA stability by protein-binding, pyrimidine-rich sequence in the 3'-untranslated region. J Biol Chem. 1999 Jan 22;274(4):2532–2538. doi: 10.1074/jbc.274.4.2532. [DOI] [PubMed] [Google Scholar]
  40. Penolazzi L., Lambertini E., Aguiari G., del Senno L., Piva R. Cis element 'decoy' against the upstream promoter of the human estrogen receptor gene. Biochim Biophys Acta. 2000 Jul 24;1492(2-3):560–567. doi: 10.1016/s0167-4781(00)00145-7. [DOI] [PubMed] [Google Scholar]
  41. Penolazzi L., Lambertini E., Aguiari G., del Senno L., Piva R. Modulation of estrogen receptor gene expression in human breast cancer cells: a decoy strategy with specific PCR-generated DNA fragments. Breast Cancer Res Treat. 1998 Jun;49(3):227–235. doi: 10.1023/a:1006060523381. [DOI] [PubMed] [Google Scholar]
  42. Russell J. E., Morales J., Makeyev A. V., Liebhaber S. A. Sequence divergence in the 3' untranslated regions of human zeta- and alpha-globin mRNAs mediates a difference in their stabilities and contributes to efficient alpha-to-zeta gene development switching. Mol Cell Biol. 1998 Apr;18(4):2173–2183. doi: 10.1128/mcb.18.4.2173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stefanovic B., Hellerbrand C., Holcik M., Briendl M., Aliebhaber S., Brenner D. A. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells. Mol Cell Biol. 1997 Sep;17(9):5201–5209. doi: 10.1128/mcb.17.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Thisted T., Lyakhov D. L., Liebhaber S. A. Optimized RNA targets of two closely related triple KH domain proteins, heterogeneous nuclear ribonucleoprotein K and alphaCP-2KL, suggest Distinct modes of RNA recognition. J Biol Chem. 2001 Feb 2;276(20):17484–17496. doi: 10.1074/jbc.M010594200. [DOI] [PubMed] [Google Scholar]
  45. Tomita S., Tomita N., Yamada T., Zhang L., Kaneda Y., Morishita R., Ogihara T., Dzau V. J., Horiuchi M. Transcription factor decoy to study the molecular mechanism of negative regulation of renin gene expression in the liver in vivo. Circ Res. 1999 May 14;84(9):1059–1066. doi: 10.1161/01.res.84.9.1059. [DOI] [PubMed] [Google Scholar]
  46. Tuerk C., Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. doi: 10.1126/science.2200121. [DOI] [PubMed] [Google Scholar]
  47. Walter B. L., Nguyen J. H., Ehrenfeld E., Semler B. L. Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements. RNA. 1999 Dec;5(12):1570–1585. doi: 10.1017/s1355838299991483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wang X., Kiledjian M., Weiss I. M., Liebhaber S. A. Detection and characterization of a 3' untranslated region ribonucleoprotein complex associated with human alpha-globin mRNA stability. Mol Cell Biol. 1995 Mar;15(3):1769–1777. doi: 10.1128/mcb.15.3.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Weiss I. M., Liebhaber S. A. Erythroid cell-specific determinants of alpha-globin mRNA stability. Mol Cell Biol. 1994 Dec;14(12):8123–8132. doi: 10.1128/mcb.14.12.8123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weiss I., Cash F. E., Coleman M. B., Pressley A., Adams J. G., Sanguansermsri T., Liebhaber S. A., Steinberg M. H. Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg) Blood. 1990 Dec 15;76(12):2630–2636. [PubMed] [Google Scholar]
  51. White R. R., Sullenger B. A., Rusconi C. P. Developing aptamers into therapeutics. J Clin Invest. 2000 Oct;106(8):929–934. doi: 10.1172/JCI11325. [DOI] [PMC free article] [PubMed] [Google Scholar]

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