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. 2002 Dec;8(12):1515–1525.

An H/ACA guide RNA directs U2 pseudouridylation at two different sites in the branchpoint recognition region in Xenopus oocytes.

Xinliang Zhao 1, Zhu-Hong Li 1, Rebecca M Terns 1, Michael P Terns 1, Yi-Tao Yu 1
PMCID: PMC1370357  PMID: 12515384

Abstract

U2 is the most extensively modified of all spliceosomal snRNAs. We previously showed that at least some of the internally modified nucleotides in U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing. Recent work from several laboratories suggests that nuclear guide RNAs facilitate U2 snRNA internal modification, including pseudouridylation and 2'-O-methylation. Here, we present a novel approach to identifying guide RNAs for U2 pseudouridylation. Several Xenopus oocyte nuclear RNAs were affinity selected with U2 snRNA substituted with 5-fluorouridine, a pseudouridylation inhibitor that sequesters pseudouridylases. One of these RNAs was sequenced and found to be a novel RNA of 134 nt. This small RNA contains an H/ACA motif and folds into a typical H/ACA RNA structure, and its authenticity as an H/ACA RNA was confirmed by immunoprecipitation analysis. The RNA contains two guide sequences for pseudouridylation (psi) of U2 snRNA at positions 34 and 44 in the branch-site recognition region, and we demonstrate that this RNA indeed guides the formation of psi34 and psi44 in U2 using a Xenopus oocyte reconstitution system. Therefore, this novel RNA was designated pugU2-34/44, for pseudouridylation guide for U2 snRNA U34 and U44. Intranuclear localization analyses indicate that pugU2-34/44 resides within the nucleoplasm rather than nucleoli or Cajal bodies where other guide RNAs have been localized. Our results clarify the mechanism of U2 snRNA pseudouridylation in Xenopus oocytes, and have interesting implications with regard to the intranuclear localization of U2 snRNA pseudouridylation.

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Selected References

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  1. Darzacq Xavier, Jády Beáta E., Verheggen Céline, Kiss Arnold M., Bertrand Edouard, Kiss Tamás. Cajal body-specific small nuclear RNAs: a novel class of 2'-O-methylation and pseudouridylation guide RNAs. EMBO J. 2002 Jun 3;21(11):2746–2756. doi: 10.1093/emboj/21.11.2746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dragon F., Pogacić V., Filipowicz W. In vitro assembly of human H/ACA small nucleolar RNPs reveals unique features of U17 and telomerase RNAs. Mol Cell Biol. 2000 May;20(9):3037–3048. doi: 10.1128/mcb.20.9.3037-3048.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Filipowicz Witold, Pogacić Vanda. Biogenesis of small nucleolar ribonucleoproteins. Curr Opin Cell Biol. 2002 Jun;14(3):319–327. doi: 10.1016/s0955-0674(02)00334-4. [DOI] [PubMed] [Google Scholar]
  4. Ganot P., Jády B. E., Bortolin M. L., Darzacq X., Kiss T. Nucleolar factors direct the 2'-O-ribose methylation and pseudouridylation of U6 spliceosomal RNA. Mol Cell Biol. 1999 Oct;19(10):6906–6917. doi: 10.1128/mcb.19.10.6906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hüttenhofer A., Kiefmann M., Meier-Ewert S., O'Brien J., Lehrach H., Bachellerie J. P., Brosius J. RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse. EMBO J. 2001 Jun 1;20(11):2943–2953. doi: 10.1093/emboj/20.11.2943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jády B. E., Kiss T. A small nucleolar guide RNA functions both in 2'-O-ribose methylation and pseudouridylation of the U5 spliceosomal RNA. EMBO J. 2001 Feb 1;20(3):541–551. doi: 10.1093/emboj/20.3.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kiss T. Small nucleolar RNA-guided post-transcriptional modification of cellular RNAs. EMBO J. 2001 Jul 16;20(14):3617–3622. doi: 10.1093/emboj/20.14.3617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Liang Xue-Hai, Xu Yu-Xin, Michaeli Shulamit. The spliced leader-associated RNA is a trypanosome-specific sn(o) RNA that has the potential to guide pseudouridine formation on the SL RNA. RNA. 2002 Feb;8(2):237–246. doi: 10.1017/s1355838202018290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Narayanan A., Lukowiak A., Jády B. E., Dragon F., Kiss T., Terns R. M., Terns M. P. Nucleolar localization signals of box H/ACA small nucleolar RNAs. EMBO J. 1999 Sep 15;18(18):5120–5130. doi: 10.1093/emboj/18.18.5120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Narayanan A., Speckmann W., Terns R., Terns M. P. Role of the box C/D motif in localization of small nucleolar RNAs to coiled bodies and nucleoli. Mol Biol Cell. 1999 Jul;10(7):2131–2147. doi: 10.1091/mbc.10.7.2131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Newby M. I., Greenbaum N. L. A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture. RNA. 2001 Jun;7(6):833–845. doi: 10.1017/s1355838201002308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Patton J. R., Jacobson M. R., Pederson T. Pseudouridine formation in U2 small nuclear RNA. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3324–3328. doi: 10.1073/pnas.91.8.3324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Peattie D. A. Direct chemical method for sequencing RNA. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1760–1764. doi: 10.1073/pnas.76.4.1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Peculis B. A., Gall J. G. Localization of the nucleolar protein NO38 in amphibian oocytes. J Cell Biol. 1992 Jan;116(1):1–14. doi: 10.1083/jcb.116.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Smith C. M., Steitz J. A. Sno storm in the nucleolus: new roles for myriad small RNPs. Cell. 1997 May 30;89(5):669–672. doi: 10.1016/s0092-8674(00)80247-0. [DOI] [PubMed] [Google Scholar]
  16. Speckmann W., Narayanan A., Terns R., Terns M. P. Nuclear retention elements of U3 small nucleolar RNA. Mol Cell Biol. 1999 Dec;19(12):8412–8421. doi: 10.1128/mcb.19.12.8412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Terns M. P., Grimm C., Lund E., Dahlberg J. E. A common maturation pathway for small nucleolar RNAs. EMBO J. 1995 Oct 2;14(19):4860–4871. doi: 10.1002/j.1460-2075.1995.tb00167.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Terns Michael P., Terns Rebecca M. Small nucleolar RNAs: versatile trans-acting molecules of ancient evolutionary origin. Gene Expr. 2002;10(1-2):17–39. [PMC free article] [PubMed] [Google Scholar]
  19. Tollervey D., Kiss T. Function and synthesis of small nucleolar RNAs. Curr Opin Cell Biol. 1997 Jun;9(3):337–342. doi: 10.1016/s0955-0674(97)80005-1. [DOI] [PubMed] [Google Scholar]
  20. Tycowski K. T., You Z. H., Graham P. J., Steitz J. A. Modification of U6 spliceosomal RNA is guided by other small RNAs. Mol Cell. 1998 Nov;2(5):629–638. doi: 10.1016/s1097-2765(00)80161-6. [DOI] [PubMed] [Google Scholar]
  21. Venema J., Tollervey D. Ribosome synthesis in Saccharomyces cerevisiae. Annu Rev Genet. 1999;33:261–311. doi: 10.1146/annurev.genet.33.1.261. [DOI] [PubMed] [Google Scholar]
  22. Weinstein L. B., Steitz J. A. Guided tours: from precursor snoRNA to functional snoRNP. Curr Opin Cell Biol. 1999 Jun;11(3):378–384. doi: 10.1016/S0955-0674(99)80053-2. [DOI] [PubMed] [Google Scholar]
  23. Wu C. H., Murphy C., Gall J. G. The Sm binding site targets U7 snRNA to coiled bodies (spheres) of amphibian oocytes. RNA. 1996 Aug;2(8):811–823. [PMC free article] [PubMed] [Google Scholar]
  24. Yu Y. T., Shu M. D., Narayanan A., Terns R. M., Terns M. P., Steitz J. A. Internal modification of U2 small nuclear (sn)RNA occurs in nucleoli of Xenopus oocytes. J Cell Biol. 2001 Mar 19;152(6):1279–1288. doi: 10.1083/jcb.152.6.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yu Y. T., Shu M. D., Steitz J. A. Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing. EMBO J. 1998 Oct 1;17(19):5783–5795. doi: 10.1093/emboj/17.19.5783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yu Y. T., Tarn W. Y., Yario T. A., Steitz J. A. More Sm snRNAs from vertebrate cells. Exp Cell Res. 1996 Dec 15;229(2):276–281. doi: 10.1006/excr.1996.0372. [DOI] [PubMed] [Google Scholar]

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