Experimental Identification of New Functional RNA

  • Thomas Dandekar
  • Kishor Sharma
Part of the Biotechnology Intelligence Unit book series (BIOIU)


Having reviewed the multitude of different regulatory RNA structures in chapter 2, the next two chapters describe experimental and theoretical approaches to identify new cases of regulatory RNA. Depending on attitude and experience, both approaches are viable routes for revealing new RNA containing functional RNA structures. The experimental approaches are sometimes less direct but are more flexible and may lead to unexpected discoveries, for instance a regulatory protein which is even more important than the regulatory RNA in the system studied. The theoretical approaches are more focused; however they are somewhat more biased as they require some preconception of at least the basic features of the RNA structure before the advantages of a systematic and direct search for suitable regulatory RNAs can be exploited. Furthermore the theoretical approach is limited by the supposition that the RNA molecule is hidden somewhere in DNA or RNA already available as a sequence. However, also theoretical searches do not necessarily require a template or even a known RNA example previously characterized to succeed (see chapter 6 for recent examples).


Hammerhead Ribozyme Upstream Open Reading Frame Oskar mRNA GCN4 mRNA SELEX Approach 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. 1.
    Madhani HD, Guthrie C. Dynamic RNA-RNA interactions in the spliceosome. Ann Rev Genet 1994; 28: 1–26.PubMedCrossRefGoogle Scholar
  2. 2.
    Beltrame M, Tollervey D. Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA. EMBO J 1992; 11: 1531–1542.PubMedGoogle Scholar
  3. 3.
    Ferrandon D, Elphick L, Nusslein-Volhard C, St Johnston D. Staufen protein associates with the 3’UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner. Cell 1994; 79122–11232.Google Scholar
  4. 4.
    Erdélyi M, Michon A-M, Guichet A, Bogucka-Glotzer J, Ephrussi A. A requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization. Nature 1995; 377: 524–527.PubMedCrossRefGoogle Scholar
  5. 5.
    Pokrywka NJ, Stephenson EC. Microtubules are a general component of mRNA localization systems in Drosophila oocytes. Dev Biol 1995; 167: 363–370.PubMedCrossRefGoogle Scholar
  6. 6.
    Melefors Ö, Hentze MW. Translational regulation by mRNA/protein interactions in eukaryotic cells: Ferritin and beyond. BioEssays 1993; 15: 85–90.CrossRefGoogle Scholar
  7. 7.
    Dandekar T, Sibbald PR. Trans-splicing of pre-mRNA is predicted to occur in a wide range of organisms including vertebrates. Nucl Acids Res 1990; 18: 4719–4726.PubMedCrossRefGoogle Scholar
  8. 8.
    Eddy SR, Durbin R. Nucl Acids Res 1994; 22: 2079–2088.PubMedCrossRefGoogle Scholar
  9. 9.
    Gray NK, Pantopoulos K, Dandekar T, Ackrell BAC, Hentze MW. Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements. Proc Natl Acad Sci USA 1996; 93: 4925–4930.PubMedCrossRefGoogle Scholar
  10. 10.
    Bufardeci E, Fabbri S, Baldi MI, Mattoccia E, Tocchini-Valentini GP. In vitro genetic analysis of the structural features of the pre-tRNA required for determination of the 3’ splice site in the intron excision reaction. EMBO 1993; 124697–4704.Google Scholar
  11. 11.
    Cech TR. Structure and mechanisms of the large catalytic RNAs: groupI and group II introns and ribonuclease P. In: Gesteland RF, Atkins JF, eds. The RNA World. Plainview, NY: Cold Spring Harbor Laboratory Press Monograph series 1993239–269.Google Scholar
  12. 12.
    Altman S. Kirsebom L, Talbot S. Recent studies of ribonuclease P. FASEB J 1993; 7: 7–14.PubMedGoogle Scholar
  13. 13.
    Purdey M. The UK epidemic of BSE: Slow virus or chronic pestizide initiated modification of the prion protein? Med Hypothesis 1996; 46: 445–454.CrossRefGoogle Scholar
  14. 14.
    Stebbins-Boaz B, Richter JD. Translational control during early development. Crit Rev Eukaryot Gene Expr 1997; 7: 73–94.PubMedGoogle Scholar
  15. 15.
    Jin L, Loyd RV. In situ hybridization: methods and applications. J Clin Lab Anal 1997; 11: 2–9.PubMedCrossRefGoogle Scholar
  16. 16.
    Kim-Ha J, Smith JL, Macdonald PM. oskar mRNA is localized to the posterior pole of the Drosophila oocyte. Cell 1991; 66: 23–35.PubMedCrossRefGoogle Scholar
  17. 17.
    Kim-Ha J, Webster PJ, Smith JL, Macdonald PM. Multiple RNA regulatory elements mediate distinct steps in the localization of oskar mRNA. Development 1993; 119: 169–178.PubMedGoogle Scholar
  18. 18.
    Serano TL, Cohen RS. A small predicted stem-loop structure mediates oocyte localization of Drosophila Kw mRNA. Development 1995; 121: 3809–3818.PubMedGoogle Scholar
  19. 19.
    Endo T, Nadal-Ginard B. Three types of mucscle-specific gene expression in fusion-blocked rat skeletal muscle cells: Translational control in EGTA-treated cells. Cell 1987; 49: 515–526.PubMedCrossRefGoogle Scholar
  20. 20.
    Werner M, Feller A, Messenguy F, Piérard A. The leader peptide of yeast gene CPAi is essential for the translational repression of its expression. Cell 1987; 49: 805–813.PubMedCrossRefGoogle Scholar
  21. 21.
    Delbecq P, Werner M, Feller A, Filipowski RK, Messenguy F, Pierard A. A segment of mRNA encoding the leader peptide of CPA1 gene confers repressin by arginine on a heterologuos yeast gene transcript. Mol Cell Biol 1994; 14: 237H - 2390CrossRefGoogle Scholar
  22. 22.
    Müller PP, Hinnebusch AG. Multiple upstream AUG codons mediate translational control of GCN4. Cell 1986; 45: 201–207.CrossRefGoogle Scholar
  23. 23.
    Garcia-Barrio MT, Naranda T, Vazquez-de-Aldana CR, Cuesta R, Hinnebusch AG, Tamame M. GCDio, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic initiation factor-3. Genes Dev 1995; 91781–1796.Google Scholar
  24. 24.
    Grant CM, Miller PF, Hinnebusch AG. Sequences 5’ of the first upstream open reading frame in GCN4 mRNA are required for efficient translational reinitiation. Nucleic Acids Res 1995; 233980–3988.Google Scholar
  25. 25.
    Geballe AP, Spaete RR, Mocarski ES. A cis-acting element within the 5’leader of a cytomegalovirus ß transcript determines kinetic class. Cell 1986; 46: 865–872.PubMedCrossRefGoogle Scholar
  26. 26.
    McGarry TJ, Lindquist S. The perferential translation of Drosophila hsp7o mRNA requires sequences in the untranslated leader. Cell 1985; 42: 903–911.PubMedCrossRefGoogle Scholar
  27. 27.
    Zähringer J, Baliga BS, Munro HN. Proc Natl Acad Sci USA 1976; 73: 857.PubMedCrossRefGoogle Scholar
  28. 28.
    Rouault TA, Hentze MW, Dancis A, Caughman W, Harford JB, Klausner RD. Influence of altered transcription on the translational control of human ferritin expression. Proc Natl. Acad. Sci USA 1987; 84: 6335–6339.CrossRefGoogle Scholar
  29. 29.
    Hentze MW, Wright Caughman S, Rouault TA, Barriocanal JG, Dancis A, Harford JB, Klausner RD. Identification of the Iron-Responsive Element for the translational regulation of human ferritin mRNA. Science 1987; 238: 1570–1573.PubMedCrossRefGoogle Scholar
  30. 30.
    Rouault TA, Hentze MW, Haile DJ et al. The iron-responsive element binding protein: A method for the affinity purification of a regulatory RNA-binding protein. Proc Natl. Acad. Sci USA 1989; 86: 5768–5772.CrossRefGoogle Scholar
  31. 31.
    Pelle R, Murphy NB. In vivo UV-crosslinking hybridization: a powerful technique for isolating RNA binding proteins. Application to trypanosome mini-exon derived RNA. Nucleic Acids Res 1993; 25: 2453–2458.CrossRefGoogle Scholar
  32. 32.
    SenGupta DJ, 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 USA 1996; 938496–8501.Google Scholar
  33. 33.
    Paillart J-C, Berthoux L, Ottmann M, Darlix J-L, Marquet R, Ehresmann B, Ehresmann C. A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis. J Virology 1996; 8348–8354.Google Scholar
  34. 34.
    Dandekar T, Tollervey D. Mutational analysis of Schizosaccharomyces pombe U4 snRNA by plasmid exchange. Yeast 1992; 8647–653.Google Scholar
  35. 35.
    Dichtl B, Tollervey D. Pop3 is essential for the activity of the RNAse MRP and RNAse P ribonucleoproteins in vivo. EMBO J 1997; 16: 417–429.PubMedCrossRefGoogle Scholar
  36. 36.
    Goossen B, Hentze MW. Mol Cell Biol 1992; 12: 1959–1966.PubMedGoogle Scholar
  37. 37.
    Dandekar T, Stripecke R, Gray NK, Goossen B, Constable A, Johansson HE, Hentze MW. Identification of a novel iron-responsive element in murine and human erythroid delta-aminolevulinic acid synthase mRNA. EMBO J 1991; 10: 1903–1909.PubMedGoogle Scholar
  38. 38.
    Dix DJ, Lin PN, McKenzie AR, Walden WE, Theil EC. The influence of the base-paired flanking region on structure and function of the iron regulatory element. J Mol Biol 1993; 231: 230–240PubMedCrossRefGoogle Scholar
  39. 39.
    Laing LG, Hall KB. A model of the iron-responsive element RNA hairpin loop structure determined from NMR and thermodynamic data. Biochemistry 1996; 35: 13586–13596.PubMedCrossRefGoogle Scholar
  40. 40.
    Doudna JA, Grosshans C, Gooding A, Kundrot CE. Crystallization of ribozymes and small RNA motifs by a sparse matrix approach. Proc Natl Acad Sci USA 1993; 90: 7829–7833.PubMedCrossRefGoogle Scholar
  41. 41.
    Dandekar T, Argos P. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy (commentary). Chemtracts 1997; in press.Google Scholar
  42. 42.
    DeRosier DJ. Who needs crystals anyway? Nature 1997; 386: 26–27.CrossRefGoogle Scholar
  43. 43.
    Turk C. Using the SELEX combinatorial chemistry process to find high affinity nucleic acid ligands to target molecules. Methods Mol Biol 1997; 67: 219–230.Google Scholar
  44. 44.
    Lohse PA, Szostak JW. Ribozyme-catalyzed amino-acid transfer reactions. Nature 1996; 381: 442–444.PubMedCrossRefGoogle Scholar
  45. 45.
    Hobbs FW. Palladium-catalyzed synthesis of alkynylamino nucleosides. A universal linker for nucleic acids. J Org Chem 1989; 54: 3420–3422.Google Scholar
  46. 46.
    Pan T, Dichtl B, Uhlenbeck O. Properties of an in vitro selected Pb“ cleavage motif. Biochemistry 1994; 339561–9565.Google Scholar
  47. 47.
    Pan T. Novel RNA substrates for the ribozyme from Bacillus subtilis ribonuclease P identified by in vitro selection. Biochemistry 1995; 34: 8458–8464.PubMedCrossRefGoogle Scholar
  48. 48.
    Baldi M, Mattocia E, Bufardeci E, Fabbri S, Tocchini-Valentini GP. Participation of the intron in the reaction catalyzed by the Xenopus tRNA splicing endonuclease. Science 1992; 255: 1390.CrossRefGoogle Scholar
  49. 49.
    Bufardeci E, Fabbri S, Baldi MI, Mattoccia E, Tocchini-Valentini GP. In vitro genetic analysis of the structural features of the pre-tRNA required for determination of the 3’ splice site in the intron excision reaction. EMBO J 1993; 124697–4704.Google Scholar
  50. 50.
    Carrara G, Calandra P, Fruscoloni P, Tocchini-Valentini GP. Two helices plus a linker: a small model substrate for eukaryotic RNase P. Proc Natl Acad Sci USA 1995; 92: 2627–2631.PubMedCrossRefGoogle Scholar
  51. 51.
    Williams KP, Ciafre S, Tocchini-Valentini GP. Selection of novel Mg++ dependent selfcleaving ribozymes. EMBO J 1995; 14: 4551–4557.PubMedGoogle Scholar
  52. 52.
    Scarabino D, Tocchini-Valentini GP. Influenece of substrate structure on cleavage by hammerhead ribozyme. FEBS Letters 1996; 383: 185–190.PubMedCrossRefGoogle Scholar
  53. 53.
    Burke DH, Gold L. RNA aptamers to the adenosine moiety of Sadenosyl methionine:structural inferences from variations on a theme and the reproducibility of SELEX. Nucleic Acids Res 1997; 25: 2020–2024.PubMedCrossRefGoogle Scholar
  54. 54.
    Beyer K, Dandekar T, Keller W. RNA-ligands selected by cleavage stimulation factor (CstF) contain distinct sequence motifs that function as downstream elements in 3’-end processing or pre-mRNA. J Biol Chem 1997; 272: 26769–26779.PubMedCrossRefGoogle Scholar
  55. 55.
    Hertel KJ, Nerschlag D, Uhlenbeck OC. Specificity of hammerhead ribozyme cleavage. EMBO J 1996; 15: 3751–3757.PubMedGoogle Scholar
  56. 56.
    Harada K, Martin SS, Frankel AD. Selection of RNA-binding peptides in vivo. Nature 1996; 380: 175–179.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg and R.G. Landes Company Georgetown, TX, U.S.A. 1998

Authors and Affiliations

  • Thomas Dandekar
    • 1
  • Kishor Sharma
    • 1
  1. 1.European MolecularBiology LaboratoryHeidelbergGermany

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