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Post-transcriptional Modification of RNAs by Artificial Box H/ACA and Box C/D RNPs

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RNA and DNA Editing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 718))

Abstract

RNA-guided RNA 2′-O-methylation and pseudouridylation are naturally occurring processes, in which guide RNAs specifically direct modifications to rRNAs or spliceosomal snRNAs in the nucleus of eukaryotic cells. Modifications can profoundly alter the properties of an RNA, thus influencing the contributions of the RNA to the cellular process in which it participates. Recently, it has been shown that, by expressing artificial guide RNAs (derived from naturally occurring guide RNAs), modifications can also be specifically introduced into other RNAs, thus offering an opportunity to study RNAs in vivo. Here, we present strategies for constructing guide RNAs and manipulating RNA modifications in the nucleus.

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References

  1. Matera, A. G., Terns, R. M., and Terns, M. P. (2007) Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs. Nat. Rev. Mol. Cell. Biol. 8, 209–20.

    Article  PubMed  CAS  Google Scholar 

  2. 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, 2746–56.

    Article  PubMed  CAS  Google Scholar 

  3. Bachellerie, J. P., Cavaille, J., and Huttenhofer, A. (2002) The expanding snoRNA world. Biochimie 84, 775–90.

    Article  PubMed  CAS  Google Scholar 

  4. Vitali, P., Royo, H., Seitz, H., Bachellerie, J. P., Huttenhofer, A., and Cavaille, J. (2003) Identification of 13 novel human modification guide RNAs. Nucleic Acids Res. 31, 6543–51.

    Article  PubMed  CAS  Google Scholar 

  5. Huttenhofer, A., Kiefmann, M., Meier-Ewert, S., O’Brien, J., Lehrach, H., Bachellerie, J. P., et al. (2001) RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse. EMBO J 20, 2943–53.

    Article  PubMed  CAS  Google Scholar 

  6. Schattner, P., Barberan-Soler, S., and Lowe, T. M. (2006) A computational screen for mammalian pseudouridylation guide H/ACA RNAs. RNA 12, 15–25.

    Article  PubMed  CAS  Google Scholar 

  7. Schattner, P., Decatur, W. A., Davis, C. A., Ares, M., Jr., Fournier, M. J., and Lowe, T. M. (2004) Genome-wide searching for ­pseudouridylation guide snoRNAs: analysis of the Saccharomyces cerevisiae genome. Nucleic Acids Res. 32, 4281–96.

    Article  PubMed  CAS  Google Scholar 

  8. Yu, Y. T., Terns, R. M., and Terns M. P. (2005) Mechanisms and Functions of RNA-guided RNA Modification. In: Fine-Tuning of RNA Functions by Modification and Editing, Springer, Berlin, pp. 223–262.

    Google Scholar 

  9. Gu, A. D., Zhou, H., Yu, C. H., and Qu, L. H. (2005) A novel experimental approach for systematic identification of box H/ACA snoRNAs from eukaryotes. Nucleic Acids Res. 33, e194.

    Article  PubMed  Google Scholar 

  10. Kiss, A. M., Jady, B. E., Bertrand, E., and Kiss, T. (2004) Human box H/ACA pseudouridylation guide RNA machinery. Mol. Cell. Biol. 24, 5797–807.

    Article  PubMed  CAS  Google Scholar 

  11. Dunbar, D. A., Wormsley, S., Lowe, T. M., and Baserga, S. J. (2000) Fibrillarin-associated box C/D small nucleolar RNAs in Trypanosoma brucei. Sequence conservation and implications for 2′-O-ribose methylation of rRNA. J Biol. Chem. 275, 14767–76.

    Article  PubMed  CAS  Google Scholar 

  12. Gaspin, C., Cavaille, J., Erauso, G., and Bachellerie, J. P. (2000) Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes. J Mol. Biol. 297, 895–906.

    Article  PubMed  CAS  Google Scholar 

  13. Omer, A. D., Lowe, T. M., Russell, A. G., Ebhardt, H., Eddy, S. R., and Dennis, P. P. (2000) Homologs of small nucleolar RNAs in Archaea. Science 288, 517–22.

    Article  PubMed  CAS  Google Scholar 

  14. Qu, L. H., Meng, Q., Zhou, H., and Chen, Y. Q. (2001) Identification of 10 novel snoRNA gene clusters from Arabidopsis thaliana. Nucleic Acids Res. 29, 1623–30.

    Article  PubMed  CAS  Google Scholar 

  15. Marker, C., Zemann, A., Terhorst, T., Kiefmann, M., Kastenmayer, J. P., Green, P., et al. (2002) Experimental RNomics: identification of 140 candidates for small non-messenger RNAs in the plant Arabidopsis thaliana. Curr. Biol. 12, 2002–13.

    Article  PubMed  CAS  Google Scholar 

  16. Tang, T. H., Bachellerie, J. P., Rozhdestvensky, T., Bortolin, M. L., Huber, H., Drungowski, M., et al. (2002) Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus. Proc. Natl. Acad. Sci. USA 99, 7536–41.

    Article  PubMed  CAS  Google Scholar 

  17. Yuan, G., Klambt, C., Bachellerie, J. P., Brosius, J., and Huttenhofer, A. (2003) RNomics in Drosophila melanogaster: identification of 66 candidates for novel non-messenger RNAs. Nucleic Acids Res. 31, 2495–507.

    Article  PubMed  CAS  Google Scholar 

  18. Balakin, A. G., Smith, L., and Fournier, M. J. (1996) The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions. Cell 86, 823–34.

    Article  PubMed  CAS  Google Scholar 

  19. Lafontaine, D. L., and Tollervey, D. (2000) Synthesis and assembly of the box C+D small nucleolar RNPs. Mol. Cell. Biol. 20, 2650–9.

    Article  PubMed  CAS  Google Scholar 

  20. Gautier, T., Berges, T., Tollervey, D., and Hurt, E. (1997) Nucleolar KKE/D repeat proteins Nop56p and Nop58p interact with Nop1p and are required for ribosome biogenesis. Mol. Cell. Biol. 17, 7088–98.

    PubMed  CAS  Google Scholar 

  21. Omer, A. D., Ziesche, S., Ebhardt, H., and Dennis, P. P. (2002) In vitroreconstitution and activity of a C/D box methylation guide ribonucleoprotein complex. Proc. Natl. Acad. Sci. USA 99, 5289–94.

    Article  PubMed  CAS  Google Scholar 

  22. Ochs, R. L., Lischwe, M. A., Spohn, W. H., and Busch, H. (1985) Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. Biol. Cell 54, 123–33.

    PubMed  CAS  Google Scholar 

  23. Galardi, S., Fatica, A., Bachi, A., Scaloni, A., Presutti, C., and Bozzoni, I. (2002) Purified box C/D snoRNPs are able to reproduce site-specific 2′-O-methylation of target RNA in vitro. Mol. Cell. Biol. 22, 6663–8.

    Article  PubMed  CAS  Google Scholar 

  24. Watkins, N. J., Segault, V., Charpentier, B., Nottrott, S., Fabrizio, P., Bachi, A., et al. (2000) A common core RNP structure shared between the small nucleoar box C/D RNPs and the spliceosomal U4 snRNP. Cell 103, 457–66.

    Article  PubMed  CAS  Google Scholar 

  25. Kuhn, J. F., Tran, E. J., and Maxwell, E. S. (2002) Archaeal ribosomal protein L7 is a functional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein. Nucleic Acids Res. 30, 931–41.

    Article  PubMed  CAS  Google Scholar 

  26. Lafontaine, D. L., and Tollervey, D. (1999) Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability. RNA 5, 455–67.

    Article  PubMed  CAS  Google Scholar 

  27. Henras, A., Henry, Y., Bousquet-Antonelli, C., Noaillac-Depeyre, J., Gelugne, J. P., and Caizergues-Ferrer, M. (1998) Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs. EMBO J 17, 7078–90.

    Article  PubMed  CAS  Google Scholar 

  28. Watkins, N. J., Gottschalk, A., Neubauer, G., Kastner, B., Fabrizio, P., Mann, M., et al. (1998) Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure. RNA 4, 1549–68.

    Article  PubMed  CAS  Google Scholar 

  29. Dragon, F., Pogacic, V., and Filipowicz, W. (2000) In vitro assembly of human H/ACA small nucleolar RNPs reveals unique features of U17 and telomerase RNAs. Mol. Cell. Biol. 20, 3037–48.

    Article  PubMed  CAS  Google Scholar 

  30. Pogacic, V., Dragon, F., and Filipowicz, W. (2000) Human H/ACA small nucleolar RNPs and telomerase share evolutionarily conserved proteins NHP2 and NOP10. Mol. Cell. Biol. 20, 9028–40.

    Article  PubMed  CAS  Google Scholar 

  31. Watanabe, Y., and Gray, M. W. (2000) Evolutionary appearance of genes encoding proteins associated with box H/ACA snoRNAs: cbf5p in Euglena gracilis, an early diverging eukaryote, and candidate Gar1p and Nop10p homologs in archaebacteria. Nucleic Acids Res. 28, 2342–52.

    Article  PubMed  CAS  Google Scholar 

  32. Rozhdestvensky, T. S., Tang, T. H., Tchirkova, I. V., Brosius, J., Bachellerie, J. P., and Huttenhofer, A. (2003) Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea. Nucleic Acids Res. 31, 869–77.

    Article  PubMed  CAS  Google Scholar 

  33. Wang, C., and Meier, U. T. (2004) Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins. EMBO J 23, 1857–67.

    Article  PubMed  CAS  Google Scholar 

  34. 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, 1077–88.

    Article  PubMed  CAS  Google Scholar 

  35. 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.

    Article  PubMed  CAS  Google Scholar 

  36. Ni, J., Tien, A. L., and Fournier, M. J. (1997) Small nucleolar RNAs direct site-specific synthesis of pseudouridine in ribosomal RNA. Cell 89, 565–73.

    Article  PubMed  CAS  Google Scholar 

  37. Bachellerie, J. P., Michot, B., Nicoloso, M., Balakin, A., Ni, J., and Fournier, M. J. (1995) Antisense snoRNAs: a family of nucleolar RNAs with long complementarities to rRNA. Trends Biochem. Sci. 20, 261–4.

    Article  PubMed  CAS  Google Scholar 

  38. Cavaille, J., Nicoloso, M., and Bachellerie, J. P. (1996) Targeted ribose methylation of RNA in vivodirected by tailored antisense RNA guides. Nature 383, 732–5.

    Article  PubMed  CAS  Google Scholar 

  39. Tollervey, D., Lehtonen, H., Jansen, R., Kern, H., and Hurt, E. C. (1993) Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly. Cell 72, 443–57.

    Article  PubMed  CAS  Google Scholar 

  40. Zebarjadian, Y., King, T., Fournier, M. J., Clarke, L., and Carbon, J. (1999) Point mutations in yeast CBF5 can abolish in vivopseudouridylation of rRNA. Mol. Cell. Biol. 19, 7461–72.

    PubMed  CAS  Google Scholar 

  41. Agris, P. F. (1996) The importance of being modified: roles of modified nucleosides and Mg2+ in RNA structure and function. Prog. Nucleic Acid Res. Mol. Biol. 53, 79–129.

    Article  PubMed  CAS  Google Scholar 

  42. Arnez, J. G., and Steitz, T. A. (1994) Crystal structure of unmodified tRNA(Gln) complexed with glutaminyl-tRNA synthetase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure. Biochemistry 33, 7560–7.

    Article  PubMed  CAS  Google Scholar 

  43. Davis, D. R. (1995) Stabilization of RNA stacking by pseudouridine. Nucleic Acids Res. 23, 5020–6.

    Article  PubMed  CAS  Google Scholar 

  44. Auffinger, P., and Westhof, E. (1997) Rules governing the orientation of the 2′-hydroxyl group in RNA. J Mol. Biol. 274, 54–63.

    Article  PubMed  CAS  Google Scholar 

  45. Auffinger, P., and Westhof, E. (1998) Hydration of RNA base pairs. J Biomol. Struct. Dyn. 16, 693–707.

    PubMed  CAS  Google Scholar 

  46. Helm, M. (2006) Post-transcriptional nucleotide modification and alternative folding of RNA. Nucleic Acids Res. 34, 721–33.

    Article  PubMed  CAS  Google Scholar 

  47. Zhao, X., and Yu, Y. T. (2008) Targeted pre-mRNA modification for gene silencing and regulation. Nat. Methods 5, 95–100.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank the members of the Yu Laboratory for discussion and inspiration. Our work was supported by grant GM62937 (to Yi-Tao Yu) from the National Institute of Health. J.K. was supported by a NIH Institutional Ruth L. Kirschstein National Research Service Award GM068411.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA

    Chao Huang, John Karijolich & Yi-Tao Yu

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  1. Chao Huang
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  2. John Karijolich
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  3. Yi-Tao Yu
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Correspondence to Yi-Tao Yu .

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Editors and Affiliations

  1. School of Medicine, Dept. Microbiology & Molecular Genetics, University of California, Irvine, Irvine, 92697, California, USA

    Ruslan Aphasizhev

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Huang, C., Karijolich, J., Yu, YT. (2011). Post-transcriptional Modification of RNAs by Artificial Box H/ACA and Box C/D RNPs. In: Aphasizhev, R. (eds) RNA and DNA Editing. Methods in Molecular Biology, vol 718. Humana Press. https://doi.org/10.1007/978-1-61779-018-8_14

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  • DOI: https://doi.org/10.1007/978-1-61779-018-8_14

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-017-1

  • Online ISBN: 978-1-61779-018-8

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