Advertisement

Experimental Rnomics

A Global Approach to Identifying Small Nuclear RNAs and Their Targets in Different Model Organisms
  • Alexander Hüttenhofer
  • Jérome Cavaillé
  • Jean-Pierre Bachellerie
Part of the Methods in Molecular Biology book series (MIMB, volume 265)

Abstract

Non-messenger RNAs (nmRNAs) play a wide and essential role in cellular functions. Computational identification of novel nmRNAs in genomes of model organisms is severely restricted owing to their lack of an open reading frame. Hence, we describe experimental approaches for their identification by generating cDNA libraries derived from nmRNAs for which we coined the term experimental RNomics. Two different procedures are introduced for cDNA library construction. First, we describe the construction of a general purpose cDNA library from sized RNA fractions. Second, we introduce a more specialized RNomics strategy employing this approach to generate a cDNA library from a specific abundant class of nmRNAs. This is illustrated using as a paradigm the two families of small nucleolar RNAs that guide modification of nucleotides in rRNAs or spliceosomal RNAs small nuclear RNAs (snRNAs) by short antisense elements complementary to the modification site. Following the identification of novel members from the class of small nuclear RNAs by experimental RNomics, we demonstrate how their target sequences in rRNAs or snRNAs can be identified.

Key Words

RNomics small non-messenger RNA small nuclear RNA 2′-O-methylation pseudouridylation cDNA library C-tailing DNA/RNA-linker 

References

  1. 1.
    Couzin, J. (2002) Breakthrough of the year: small RNAs make big splash. Science 298, 2296–2297.PubMedCrossRefGoogle Scholar
  2. 2.
    Dennis, C. (2002) Small RNAs: the genome’s guiding hand? Nature 420, 732.PubMedCrossRefGoogle Scholar
  3. 3.
    Dennis, C. (2002) The brave new world of RNA. Nature 418, 122–124.PubMedCrossRefGoogle Scholar
  4. 4.
    Huttenhofer, A., Brosius, J., and Bachellerie, J. P. (2002) RNomics: identification and function of small, non-messenger RNAs. Curr. Opin. Chem. Biol. 6, 835–843.PubMedCrossRefGoogle Scholar
  5. 5.
    Storz, G. (2002) An expanding universe of noncoding RNAs. Science 296, 1260–1263.PubMedCrossRefGoogle Scholar
  6. 6.
    Gottesman, S. (2002) Stealth regulation: biological circuits with small RNA switches. Genes Dev. 16, 2829–2842.PubMedCrossRefGoogle Scholar
  7. 7.
    Tuschl, T. (2002) Expanding small RNA interference. Nat. Biotechnol. 20, 446–448.PubMedCrossRefGoogle Scholar
  8. 8.
    Tuschl, T. (2003) Functional genomics: RNA sets the standard. Nature 421, 220, 221.PubMedCrossRefGoogle Scholar
  9. 9.
    Ambros, V. (2001) microRNAs: tiny regulators with great potential. Cell 107, 823–826.PubMedCrossRefGoogle Scholar
  10. 10.
    Bachellerie, J. P., Cavaille, J., and Huttenhofer, A. (2002) The expanding snoRNA world. Biochimie 84, 775–790.PubMedCrossRefGoogle Scholar
  11. 11.
    Kiss, T. (2002) Small nucleolar RNAs: an abundant group of noncoding RNAs with diverse cellular functions. Cell 109, 145–148.PubMedCrossRefGoogle Scholar
  12. 12.
    Lau, N. C., Lim, L. P., Weinstein, E. G., and Bartel, D. P. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862.PubMedCrossRefGoogle Scholar
  13. 13.
    Lee, R. C. and Ambros, V. (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862–864.PubMedCrossRefGoogle Scholar
  14. 14.
    Rhoades, M. W., Reinhart, B. J., Lim, L. P., Burge, C. B., Bartel, B., and Bartel, D. P. (2002) Prediction of plant microRNA targets. Cell 110, 513–520.PubMedCrossRefGoogle Scholar
  15. 15.
    Volpe, T. A., Kidner, C., Hall, I. M., Teng, G., Grewal, S. I., and Martienssen, R. A. (2002) Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833–1837.PubMedCrossRefGoogle Scholar
  16. 16.
    Huttenhofer, A., Kiefmann, M., Meier-Ewert, S., O’Brien, J., Lehrach, H., Bachellerie, J. P., and Brosius, J. (2001) RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse. EMBP J. 20, 2943–2953.CrossRefGoogle Scholar
  17. 17.
    Marker, C., Zemann, A., Terhorst, T., Kiefmann, M., Kastenmayer, J. P., Green, P., Bachellerie, J. P., Brosius, J., and Huttenhofer, A. (2002) Experimental RNomics: identification of 140 candidates for small non-messenger RNAs in the plant Arabidopsis thaliana. Curr. Biol. 12, 2002–2013.PubMedCrossRefGoogle Scholar
  18. 18.
    Tang, T. H., Bachellerie, J. P., Rozhdestvensky, T., Bortolin, M. L., Huber, H., Drungowski, M., Elge, T., Brosius, J., and Huttenhofer, A. (2002) Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc. Natl Acad. Sci. USA 99, 7536–7541.PubMedCrossRefGoogle Scholar
  19. 19.
    Schmitt, A. O., Herwig, R., Meier-Ewert, S., and Lehrach, H. (1999) High density cDNA grids for hybridization fingerprinting experiments, in PCR Applications: Protocols for Functional Genomics (Innis, M. A., Gelfand, D. H., and Sninsky, J. J., eds.), Academic, San Diego, pp. 457–472.Google Scholar
  20. 20.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  21. 21.
    Maier, E., Meier-Ewert, S., Ahmadi, A. R., Curtis, J., and Lehrach, H. (1994) Application of robotic technology to automated sequence fingerprint analysis by oligonucleotide hybridisation. J. Biotechnol. 35, 191–203.PubMedCrossRefGoogle Scholar
  22. 22.
    Kiss-Laszlo, Z., Henry, Y., Bachellerie, J. P., Caizergues-Ferrer, M., and Kiss, T. (1996) Site-specific ribose methylation of preribosomal RNA: a novel function for small nucleolar RNAs. Cell 85, 1077–1088.PubMedCrossRefGoogle Scholar
  23. 23.
    Cavaille, J., Vitali, P., Basyuk, E., Huttenhofer, A., and Bachellerie, J. P. (2001) A novel brain-specific box C/D small nucleolar RNA processed from tandemly repeated introns of a noncoding RNA gene in rats. J. Biol. Chem. 276, 26,374–26,383.PubMedCrossRefGoogle Scholar
  24. 24.
    Zuker, M. (1994) Prediction of RNA secondary structure by energy minimization. Methods Mol. Biol. 25, 267–294.PubMedGoogle Scholar
  25. 25.
    Bachellerie, J. P. and Cavaille, J. (1997) Guiding ribose methylation of rRNA. Trends Biochem. Sci. 22, 257–261.PubMedCrossRefGoogle Scholar
  26. 26.
    Kiss-Laszlo, Z., Henry, Y., and Kiss, T. (1998) Sequence and structural elements of methylation guide snoRNAs essential for site-specific ribose methylation of pre-rRNA. EMBO J. 17, 797–807.PubMedCrossRefGoogle Scholar
  27. 27.
    Darzacq, X., Jady, B. E., Verheggen, C., Kiss, A. M., Bertrand, E., and Kiss, T. (2002) Cajal body-specific small nuclear RNAs: a novel class of 2′-O-methylation and pseudouridylation guide RNAs. EMBO J. 21, 2746–2756.PubMedCrossRefGoogle Scholar
  28. 28.
    Ganot, P., Caizergues-Ferrer, M., and Kiss, T. (1997) The family of box ACA small nucleolar RNAs is defined by an evolutionarily conserved secondary structure and ubiquitous sequence elements essential for RNA accumulation. Genes Dev. 11, 941–956.PubMedCrossRefGoogle Scholar
  29. 29.
    Cavaille, J. and Bachellerie, J. P. (1998) SnoRNA-guided ribose methylation of rRNA: structural features of the guide RNA duplex influencing the extent of the reaction. Nucleic Acids Res. 26, 1576–1587.PubMedCrossRefGoogle Scholar
  30. 30.
    Maden, B. E. (1990) The numerous modified nucleotides in eukaryotic ribosomal RNA. Prog. Nucleic Acid Res. Mol. Biol. 39, 241–303.PubMedCrossRefGoogle Scholar
  31. 31.
    Ofengand, J. and Bakin, A. (1997) Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts. J. Mol. Biol. 266, 246–268.PubMedCrossRefGoogle Scholar
  32. 32.
    Massenet, S., Mougin, A., and Branlant, C. (1998) Posttranscriptional modifications in the U snRNAs, in Modification and Editing of RNA: The Alteration of RNA Structure and Function (Grosjean, H. and Benne, R. E., eds.), ASM Press, Washington, DC.Google Scholar
  33. 33.
    Maden, B. E., Corbett, M. E., Heeney, P. A., Pugh, K., and Ajuh, P. M. (1995) Classical and novel approaches to the detection and localization of the numerous modified nucleotides in eukaryotic ribosomal RNA. Biochimie 77, 22–29.PubMedCrossRefGoogle Scholar
  34. 34.
    Ganot, P., Bortolin, M. L., and Kiss, T. (1997) Site-specific pseudouridine formation in preribosomal RNA is guided by small nucleolar RNAs. Cell 89, 799–809.PubMedCrossRefGoogle Scholar
  35. 35.
    Ofengand, J. and Rudd, K. (2000) Bacterial, archaea, and organellar RNA pseudouridines and methylated nucleosides and their enzymes, in Ribosome: Structure, Function, Antibiotics, and Cellular Interactions (Garrett, R., Douthwaite, S., Liljas, A., Matheson, A., Moore, P. B., and Noller, H. E., eds.), ASM Press, Washington, DC.Google Scholar
  36. 36.
    Ofengand, J. and Fournier, M. J. (1998) The pseudouridine residues of rRNA: number, location, biosynthesis and function, in Modification and Editing of RNA: The Alteration of RNA Structure and Function (Grosjean, H. and Benne, R. E., eds.), ASM Press, Washington, DC.Google Scholar
  37. 37.
    Vidovic, I., Nottrott, S., Hartmuth, K., Luhrmann, R., and Ficner, R. (2000) Crystal structure of the spliceosomal 15.5kD protein bound to a U4 snRNA fragment. Mol. Cell 6, 1331–1342.PubMedCrossRefGoogle Scholar
  38. 38.
    Darzacq, X. and Kiss, T. (2000) Processing of intron-encoded box C/D small nucleolar RNAs lacking a 5′, 3′-terminal stem structure. Mol. Cell. Biol. 20, 4522–4531.PubMedCrossRefGoogle Scholar
  39. 39.
    Kiss, A. M., Jady, B. E., Darzacq, X., Verheggen, C., Bertrand, E., and Kiss, T. (2002) A Cajal body-specific pseudouridylation guide RNA is composed of two box H/ACA snoRNA-like domains. Nucleic Acids Res. 30, 4643–4649.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Alexander Hüttenhofer
    • 1
  • Jérome Cavaillé
    • 2
  • Jean-Pierre Bachellerie
    • 2
  1. 1.Institut für Molekularbiologie, Abt. Funktionelle GenomikUniversität InnsbruckInnsbruckAustria
  2. 2.Laboratoire de Biologie Moléculaire Eucaryote du CNRSUniversité Paul SabatierToulouseFrance

Personalised recommendations