Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

RNA logoLink to RNA
. 2001 Jun;7(6):833–845. doi: 10.1017/s1355838201002308

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture.

M I Newby 1, N L Greenbaum 1
PMCID: PMC1370140  PMID: 11424937

Abstract

The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 2'OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (psi) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of psi at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of psi also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex deltaG degrees 37. These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.

Full Text

The Full Text of this article is available as a PDF (388.6 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arnez J. G., Steitz T. A. 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. 1994 Jun 21;33(24):7560–7567. doi: 10.1021/bi00190a008. [DOI] [PubMed] [Google Scholar]
  2. Bakin A., Lane B. G., Ofengand J. Clustering of pseudouridine residues around the peptidyltransferase center of yeast cytoplasmic and mitochondrial ribosomes. Biochemistry. 1994 Nov 15;33(45):13475–13483. doi: 10.1021/bi00249a036. [DOI] [PubMed] [Google Scholar]
  3. Behrens S. E., Tyc K., Kastner B., Reichelt J., Lührmann R. Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions. Mol Cell Biol. 1993 Jan;13(1):307–319. doi: 10.1128/mcb.13.1.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bevilacqua P. C., Turner D. H. Comparison of binding of mixed ribose-deoxyribose analogues of CUCU to a ribozyme and to GGAGAA by equilibrium dialysis: evidence for ribozyme specific interactions with 2' OH groups. Biochemistry. 1991 Nov 5;30(44):10632–10640. doi: 10.1021/bi00108a005. [DOI] [PubMed] [Google Scholar]
  5. Borer P. N., Lin Y., Wang S., Roggenbuck M. W., Gott J. M., Uhlenbeck O. C., Pelczer I. Proton NMR and structural features of a 24-nucleotide RNA hairpin. Biochemistry. 1995 May 16;34(19):6488–6503. doi: 10.1021/bi00019a030. [DOI] [PubMed] [Google Scholar]
  6. Callihan D., West J., Kumar S., Schweitzer B. I., Logan T. M. Simple, distortion-free homonuclear spectra of peptides and nucleic acids in water using excitation sculpting. J Magn Reson B. 1996 Jul;112(1):82–85. doi: 10.1006/jmrb.1996.0114. [DOI] [PubMed] [Google Scholar]
  7. Chu V. T., Liu Q., Podar M., Perlman P. S., Pyle A. M. More than one way to splice an RNA: branching without a bulge and splicing without branching in group II introns. RNA. 1998 Oct;4(10):1186–1202. doi: 10.1017/s1355838298980724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davis D. R., Poulter C. D. 1H-15N NMR studies of Escherichia coli tRNA(Phe) from hisT mutants: a structural role for pseudouridine. Biochemistry. 1991 Apr 30;30(17):4223–4231. doi: 10.1021/bi00231a017. [DOI] [PubMed] [Google Scholar]
  9. Davis D. R. Stabilization of RNA stacking by pseudouridine. Nucleic Acids Res. 1995 Dec 25;23(24):5020–5026. doi: 10.1093/nar/23.24.5020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Durant P. C., Davis D. R. Stabilization of the anticodon stem-loop of tRNALys,3 by an A+-C base-pair and by pseudouridine. J Mol Biol. 1999 Jan 8;285(1):115–131. doi: 10.1006/jmbi.1998.2297. [DOI] [PubMed] [Google Scholar]
  11. Foster P. G., Huang L., Santi D. V., Stroud R. M. The structural basis for tRNA recognition and pseudouridine formation by pseudouridine synthase I. Nat Struct Biol. 2000 Jan;7(1):23–27. doi: 10.1038/71219. [DOI] [PubMed] [Google Scholar]
  12. Freier S. M., Kierzek R., Jaeger J. A., Sugimoto N., Caruthers M. H., Neilson T., Turner D. H. Improved free-energy parameters for predictions of RNA duplex stability. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9373–9377. doi: 10.1073/pnas.83.24.9373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greenbaum N. L., Radhakrishnan I., Hirsh D., Patel D. J. Determination of the folding topology of the SL1 RNA from Caenorhabditis elegans by multidimensional heteronuclear NMR. J Mol Biol. 1995 Sep 22;252(3):314–327. doi: 10.1006/jmbi.1995.0499. [DOI] [PubMed] [Google Scholar]
  14. Greenbaum N. L., Radhakrishnan I., Patel D. J., Hirsh D. Solution structure of the donor site of a trans-splicing RNA. Structure. 1996 Jun 15;4(6):725–733. doi: 10.1016/s0969-2126(96)00078-0. [DOI] [PubMed] [Google Scholar]
  15. Gu J., Patton J. R., Shimba S., Reddy R. Localization of modified nucleotides in Schizosaccharomyces pombe spliceosomal small nuclear RNAs: modified nucleotides are clustered in functionally important regions. RNA. 1996 Sep;2(9):909–918. [PMC free article] [PubMed] [Google Scholar]
  16. Hall K. B., McLaughlin L. W. Properties of a U1/mRNA 5' splice site duplex containing pseudouridine as measured by thermodynamic and NMR methods. Biochemistry. 1991 Feb 19;30(7):1795–1801. doi: 10.1021/bi00221a010. [DOI] [PubMed] [Google Scholar]
  17. Hall K. B., McLaughlin L. W. Properties of pseudouridine N1 imino protons located in the major groove of an A-form RNA duplex. Nucleic Acids Res. 1992 Apr 25;20(8):1883–1889. doi: 10.1093/nar/20.8.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kieft J. S., Tinoco I., Jr Solution structure of a metal-binding site in the major groove of RNA complexed with cobalt (III) hexammine. Structure. 1997 May 15;5(5):713–721. doi: 10.1016/s0969-2126(97)00225-6. [DOI] [PubMed] [Google Scholar]
  19. Lee C. H., Tinoco I., Jr Studies of the conformation of modified dinucleoside phosphates containing 1,N6-ethenoadenosine and 2'-O-methylcytidine by 360-MHz 1H nuclear magnetic resonance spectroscopy. Investigation of the solution conformations of dinucleoside phosphates. Biochemistry. 1977 Dec 13;16(25):5403–5414. doi: 10.1021/bi00644a001. [DOI] [PubMed] [Google Scholar]
  20. Liu Q., Green J. B., Khodadadi A., Haeberli P., Beigelman L., Pyle A. M. Branch-site selection in a group II intron mediated by active recognition of the adenine amino group and steric exclusion of non-adenine functionalities. J Mol Biol. 1997 Mar 21;267(1):163–171. doi: 10.1006/jmbi.1996.0845. [DOI] [PubMed] [Google Scholar]
  21. Madhani H. D., Guthrie C. Randomization-selection analysis of snRNAs in vivo: evidence for a tertiary interaction in the spliceosome. Genes Dev. 1994 May 1;8(9):1071–1086. doi: 10.1101/gad.8.9.1071. [DOI] [PubMed] [Google Scholar]
  22. Massenet S., Motorin Y., Lafontaine D. L., Hurt E. C., Grosjean H., Branlant C. Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA. Mol Cell Biol. 1999 Mar;19(3):2142–2154. doi: 10.1128/mcb.19.3.2142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McPheeters D. S., Abelson J. Mutational analysis of the yeast U2 snRNA suggests a structural similarity to the catalytic core of group I introns. Cell. 1992 Nov 27;71(5):819–831. doi: 10.1016/0092-8674(92)90557-s. [DOI] [PubMed] [Google Scholar]
  24. Meroueh M., Grohar P. J., Qiu J., SantaLucia J., Jr, Scaringe S. A., Chow C. S. Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA. Nucleic Acids Res. 2000 May 15;28(10):2075–2083. doi: 10.1093/nar/28.10.2075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Patel D. J., Hilbers C. W. Proton nuclear magnetic resonance investigations of fraying in double-stranded d-ApTpGpCpApT in H2O solution. Biochemistry. 1975 Jun 17;14(12):2651–2656. doi: 10.1021/bi00683a014. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Portmann S., Grimm S., Workman C., Usman N., Egli M. Crystal structures of an A-form duplex with single-adenosine bulges and a conformational basis for site-specific RNA self-cleavage. Chem Biol. 1996 Mar;3(3):173–184. doi: 10.1016/s1074-5521(96)90260-4. [DOI] [PubMed] [Google Scholar]
  28. Puglisi J. D., Tan R., Calnan B. J., Frankel A. D., Williamson J. R. Conformation of the TAR RNA-arginine complex by NMR spectroscopy. Science. 1992 Jul 3;257(5066):76–80. doi: 10.1126/science.1621097. [DOI] [PubMed] [Google Scholar]
  29. Puglisi J. D., Tinoco I., Jr Absorbance melting curves of RNA. Methods Enzymol. 1989;180:304–325. doi: 10.1016/0076-6879(89)80108-9. [DOI] [PubMed] [Google Scholar]
  30. Query C. C., Strobel S. A., Sharp P. A. Three recognition events at the branch-site adenine. EMBO J. 1996 Mar 15;15(6):1392–1402. [PMC free article] [PubMed] [Google Scholar]
  31. Scaringe S. A. Advanced 5'-silyl-2'-orthoester approach to RNA oligonucleotide synthesis. Methods Enzymol. 2000;317:3–18. doi: 10.1016/s0076-6879(00)17003-x. [DOI] [PubMed] [Google Scholar]
  32. Smith J. S., Nikonowicz E. P. NMR structure and dynamics of an RNA motif common to the spliceosome branch-point helix and the RNA-binding site for phage GA coat protein. Biochemistry. 1998 Sep 29;37(39):13486–13498. doi: 10.1021/bi981558a. [DOI] [PubMed] [Google Scholar]
  33. Thiviyanathan V., Guliaev A. B., Leontis N. B., Gorenstein D. G. Solution conformation of a bulged adenosine base in an RNA duplex by relaxation matrix refinement. J Mol Biol. 2000 Jul 28;300(5):1143–1154. doi: 10.1006/jmbi.2000.3931. [DOI] [PubMed] [Google Scholar]
  34. Varani L., Hasegawa M., Spillantini M. G., Smith M. J., Murrell J. R., Ghetti B., Klug A., Goedert M., Varani G. Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and parkinsonism linked to chromosome 17. Proc Natl Acad Sci U S A. 1999 Jul 6;96(14):8229–8234. doi: 10.1073/pnas.96.14.8229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Westman E., Strömberg R. Removal of t-butyldimethylsilyl protection in RNA-synthesis. Triethylamine trihydrofluoride (TEA, 3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF). Nucleic Acids Res. 1994 Jun 25;22(12):2430–2431. doi: 10.1093/nar/22.12.2430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wu J., Manley J. L. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 1989 Oct;3(10):1553–1561. doi: 10.1101/gad.3.10.1553. [DOI] [PubMed] [Google Scholar]
  37. Yarian C. S., Basti M. M., Cain R. J., Ansari G., Guenther R. H., Sochacka E., Czerwinska G., Malkiewicz A., Agris P. F. Structural and functional roles of the N1- and N3-protons of psi at tRNA's position 39. Nucleic Acids Res. 1999 Sep 1;27(17):3543–3549. doi: 10.1093/nar/27.17.3543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES