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. 1995 Dec;69(12):8027–8034. doi: 10.1128/jvi.69.12.8027-8034.1995

In vitro selection of RNA ligands for the ribosomal L22 protein associated with Epstein-Barr virus-expressed RNA by using randomized and cDNA-derived RNA libraries.

M Dobbelstein 1, T Shenk 1
PMCID: PMC189748  PMID: 7494316

Abstract

The Epstein-Barr virus (EBV)-expressed RNA 1 (EBER1) associates tightly with the ribosomal protein L22. We determined the general requirements for an RNA to bind L22 in a SELEX experiment, selecting RNA ligands for L22 from a randomized pool of RNA sequences by using an L22-glutathione S-transferase fusion protein. The selected sequences all contained a stem-loop motif similar to that of the region of EBER1 previously shown to interact with L22. The nucleotides were highly conserved at three positions within the stem-loop and identical to the corresponding nucleotides in EBER1. Two independent binding sites for L22 could be identified in EBER1, and mobility shift assays indicated that two L22 molecules can interact with EBER1 simultaneously. To search for a cellular L22 ligand, we constructed a SELEX library from cDNA fragments derived from RNA that was coimmunoprecipitated with L22 from an EBV-negative whole-cell lysate. After four rounds of selection and amplification, most of the clones that were obtained overlapped a sequence corresponding to the stem-loop between nucleotides 302 and 317 in human 28S ribosomal RNA. This stem-loop fulfills the criteria for optimal binding to L22 that were defined by SELEX, suggesting that human 28S ribosomal RNA is likely to be a cellular L22 ligand. Additional L22 binding sites were found in 28S ribosomal RNA, as well as within 18S ribosomal RNA and in RNA segments not present in sequence databases. The methodology described for the conversion of a preselected cellular RNA pool into a SELEX library might be generally applicable to other proteins for the identification of cellular RNA ligands.

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Selected References

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  1. Chan Y. L., Wool I. G. The primary structure of rat ribosomal protein L22. Biochim Biophys Acta. 1995 Jan 2;1260(1):113–115. doi: 10.1016/0167-4781(94)00193-7. [DOI] [PubMed] [Google Scholar]
  2. Clark C. G., Tague B. W., Ware V. C., Gerbi S. A. Xenopus laevis 28S ribosomal RNA: a secondary structure model and its evolutionary and functional implications. Nucleic Acids Res. 1984 Aug 10;12(15):6197–6220. doi: 10.1093/nar/12.15.6197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dolecki G. J., Lum R., Humphreys T. A gene expressed in the endoderm of the sea urchin embryo. DNA. 1988 Nov;7(9):637–643. doi: 10.1089/dna.1988.7.637. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Froussard P. A random-PCR method (rPCR) to construct whole cDNA library from low amounts of RNA. Nucleic Acids Res. 1992 Jun 11;20(11):2900–2900. doi: 10.1093/nar/20.11.2900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Froussard P. rPCR: a powerful tool for random amplification of whole RNA sequences. PCR Methods Appl. 1993 Feb;2(3):185–190. doi: 10.1101/gr.2.3.185. [DOI] [PubMed] [Google Scholar]
  7. Gao F. B., Carson C. C., Levine T., Keene J. D. Selection of a subset of mRNAs from combinatorial 3' untranslated region libraries using neuronal RNA-binding protein Hel-N1. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11207–11211. doi: 10.1073/pnas.91.23.11207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gilligan K., Rajadurai P., Resnick L., Raab-Traub N. Epstein-Barr virus small nuclear RNAs are not expressed in permissively infected cells in AIDS-associated leukoplakia. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8790–8794. doi: 10.1073/pnas.87.22.8790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Glickman J. N., Howe J. G., Steitz J. A. Structural analyses of EBER1 and EBER2 ribonucleoprotein particles present in Epstein-Barr virus-infected cells. J Virol. 1988 Mar;62(3):902–911. doi: 10.1128/jvi.62.3.902-911.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gonzalez I. L., Gorski J. L., Campen T. J., Dorney D. J., Erickson J. M., Sylvester J. E., Schmickel R. D. Variation among human 28S ribosomal RNA genes. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7666–7670. doi: 10.1073/pnas.82.22.7666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Greenspan J. S., Greenspan D., Lennette E. T., Abrams D. I., Conant M. A., Petersen V., Freese U. K. Replication of Epstein-Barr virus within the epithelial cells of oral "hairy" leukoplakia, an AIDS-associated lesion. N Engl J Med. 1985 Dec 19;313(25):1564–1571. doi: 10.1056/NEJM198512193132502. [DOI] [PubMed] [Google Scholar]
  12. Howe J. G., Shu M. D. Isolation and characterization of the genes for two small RNAs of herpesvirus papio and their comparison with Epstein-Barr virus-encoded EBER RNAs. J Virol. 1988 Aug;62(8):2790–2798. doi: 10.1128/jvi.62.8.2790-2798.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klein G. Epstein-Barr virus strategy in normal and neoplastic B cells. Cell. 1994 Jun 17;77(6):791–793. doi: 10.1016/0092-8674(94)90125-2. [DOI] [PubMed] [Google Scholar]
  14. Lavergne J. P., Conquet F., Reboud J. P., Reboud A. M. Role of acidic phosphoproteins in the partial reconstitution of the active 60 S ribosomal subunit. FEBS Lett. 1987 May 25;216(1):83–88. doi: 10.1016/0014-5793(87)80761-5. [DOI] [PubMed] [Google Scholar]
  15. Lerner M. R., Andrews N. C., Miller G., Steitz J. A. Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A. 1981 Feb;78(2):805–809. doi: 10.1073/pnas.78.2.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Michot B., Hassouna N., Bachellerie J. P. Secondary structure of mouse 28S rRNA and general model for the folding of the large rRNA in eukaryotes. Nucleic Acids Res. 1984 May 25;12(10):4259–4279. doi: 10.1093/nar/12.10.4259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Myer V. E., Lee S. I., Steitz J. A. Viral small nuclear ribonucleoproteins bind a protein implicated in messenger RNA destabilization. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1296–1300. doi: 10.1073/pnas.89.4.1296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schneider D., Gold L., Platt T. Selective enrichment of RNA species for tight binding to Escherichia coli rho factor. FASEB J. 1993 Jan;7(1):201–207. doi: 10.1096/fasebj.7.1.7678562. [DOI] [PubMed] [Google Scholar]
  20. Steinitz M., Klein G. Comparison between growth characteristics of an Epstein--Barr virus (EBV)-genome-negative lymphoma line and its EBV-converted subline in vitro. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3518–3520. doi: 10.1073/pnas.72.9.3518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Steitz J. A. Immunoprecipitation of ribonucleoproteins using autoantibodies. Methods Enzymol. 1989;180:468–481. doi: 10.1016/0076-6879(89)80118-1. [DOI] [PubMed] [Google Scholar]
  22. Swaminathan S., Huneycutt B. S., Reiss C. S., Kieff E. Epstein-Barr virus-encoded small RNAs (EBERs) do not modulate interferon effects in infected lymphocytes. J Virol. 1992 Aug;66(8):5133–5136. doi: 10.1128/jvi.66.8.5133-5136.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Swaminathan S., Tomkinson B., Kieff E. Recombinant Epstein-Barr virus with small RNA (EBER) genes deleted transforms lymphocytes and replicates in vitro. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1546–1550. doi: 10.1073/pnas.88.4.1546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Toczyski D. P., Matera A. G., Ward D. C., Steitz J. A. The Epstein-Barr virus (EBV) small RNA EBER1 binds and relocalizes ribosomal protein L22 in EBV-infected human B lymphocytes. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3463–3467. doi: 10.1073/pnas.91.8.3463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Toczyski D. P., Steitz J. A. EAP, a highly conserved cellular protein associated with Epstein-Barr virus small RNAs (EBERs). EMBO J. 1991 Feb;10(2):459–466. doi: 10.1002/j.1460-2075.1991.tb07968.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Toczyski D. P., Steitz J. A. The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1. Mol Cell Biol. 1993 Jan;13(1):703–710. doi: 10.1128/mcb.13.1.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Tuerk C., MacDougal-Waugh S. In vitro evolution of functional nucleic acids: high-affinity RNA ligands of HIV-1 proteins. Gene. 1993 Dec 27;137(1):33–39. doi: 10.1016/0378-1119(93)90248-2. [DOI] [PubMed] [Google Scholar]
  29. Walling D. M., Clark N. M., Markovitz D. M., Frank T. S., Braun D. K., Eisenberg E., Krutchkoff D. J., Felix D. H., Raab-Traub N. Epstein-Barr virus coinfection and recombination in non-human immunodeficiency virus-associated oral hairy leukoplakia. J Infect Dis. 1995 May;171(5):1122–1130. doi: 10.1093/infdis/171.5.1122. [DOI] [PubMed] [Google Scholar]
  30. Walling D. M., Edmiston S. N., Sixbey J. W., Abdel-Hamid M., Resnick L., Raab-Traub N. Coinfection with multiple strains of the Epstein-Barr virus in human immunodeficiency virus-associated hairy leukoplakia. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6560–6564. doi: 10.1073/pnas.89.14.6560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wassarman D. A., Lee S. I., Steitz J. A. Nucleotide sequence of HSUR 5 RNA from herpesvirus saimiri. Nucleic Acids Res. 1989 Feb 11;17(3):1258–1258. doi: 10.1093/nar/17.3.1258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wool I. G. The structure and function of eukaryotic ribosomes. Annu Rev Biochem. 1979;48:719–754. doi: 10.1146/annurev.bi.48.070179.003443. [DOI] [PubMed] [Google Scholar]
  33. Zuker M. Prediction of RNA secondary structure by energy minimization. Methods Mol Biol. 1994;25:267–294. doi: 10.1385/0-89603-276-0:267. [DOI] [PubMed] [Google Scholar]
  34. van Santen V., Cheung A., Kieff E. Epstein-Barr virus RNA VII: size and direction of transcription of virus-specified cytoplasmic RNAs in a transformed cell line. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1930–1934. doi: 10.1073/pnas.78.3.1930. [DOI] [PMC free article] [PubMed] [Google Scholar]

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