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U2–U6 RNA folding reveals a group II intron-like domain and a four-helix junction

Nature Structural & Molecular Biology volume 11, pages 1237–1242 (2004)Cite this article

A Corrigendum to this article was published on 01 January 2005

Abstract

Intron removal in nuclear precursor mRNA is catalyzed through two transesterification reactions by a multi-megaDalton ribonucleoprotein machine called the spliceosome. A complex between U2 and U6 small nuclear RNAs is a core component of the spliceosome. Here we present an NMR structural analysis of a protein-free U2–U6 complex from Saccharomyces cerevisiae. The observed folding of the U2–U6 complex is a four-helix junction, in which the catalytically important AGC triad base-pairs only within U6 and not with U2. The base-pairing of the AGC triad extends the U6 intramolecular stem-loop (U6 ISL), and the NMR structure of this extended U6 ISL reveals structural similarities with domain 5 of group II self-splicing introns. The observed conformation of the four-helix junction could be relevant to the first, but not the second, step of splicing and may help to position the U6 ISL adjacent to the 5′ splice site.

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Figure 1: Sequence of the U2–U6 complex from Saccharomyces cerevisiae.
Figure 2: NMR evidence for secondary structure of U2–U6 reveals a four-helix junction conformation.
Figure 3: NMR structure of the extended U6 ISL, as observed in the context of the U2–U6 complex.
Figure 4: Structural comparison of the extended U6 ISL and D5.
Figure 5: Model for U2–U6 conformational change after first step of splicing.

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References

  1. Nilsen, T.W. RNA-RNA Interactions in Nuclear Pre-mRNA Splicing (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998).

    Google Scholar 

  2. Brow, D.A. Allosteric cascade of spliceosome activation. Annu. Rev. Genet. 36, 333–360 (2002).

    Article  CAS  Google Scholar 

  3. Jacquier, A. Self-splicing group II and nuclear pre-mRNA introns: how similar are they? Trends Biochem. Sci. 15, 351–354 (1990).

    Article  CAS  Google Scholar 

  4. Moore, M.J. & Sharp, P.A. Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing. Nature 365, 364–368 (1993).

    Article  CAS  Google Scholar 

  5. Padgett, R.A., Podar, M., Boulanger, S.C. & Perlman, P.S. The stereochemical course of group II intron self-splicing. Science 266, 1685–1688 (1994).

    Article  CAS  Google Scholar 

  6. Villa, T., Pleiss, J.A. & Guthrie, C. Spliceosomal snRNAs: Mg(2+)-dependent chemistry at the catalytic core? Cell 109, 149–152 (2002).

    Article  CAS  Google Scholar 

  7. Valadkhan, S. & Manley, J.L. Splicing-related catalysis by protein-free snRNAs. Nature 413, 701–707 (2001).

    Article  CAS  Google Scholar 

  8. Valadkhan, S. & Manley, J.L. Characterization of the catalytic activity of U2 and U6 snRNAs. RNA 9, 892–904 (2003).

    Article  CAS  Google Scholar 

  9. Madhani, H.D. & Guthrie, C. A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome. Cell 71, 803–817 (1992).

    Article  CAS  Google Scholar 

  10. Wu, J. & Manley, J.L. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 3, 1553–1561 (1989).

    Article  CAS  Google Scholar 

  11. Parker, R., Siliciano, P.G. & Guthrie, C. Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell 49, 229–239 (1987).

    Article  CAS  Google Scholar 

  12. Newby, M.I. & Greenbaum, N.L. Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine. Nat. Struct. Biol. 9, 958–965 (2002).

    Article  CAS  Google Scholar 

  13. Lesser, C.F. & Guthrie, C. Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science 262, 1982–1988 (1993).

    Article  CAS  Google Scholar 

  14. Kandels-Lewis, S. & Seraphin, B. Involvement of U6 snRNA in 5′ splice site selection. Science 262, 2035–2039 (1993).

    Article  CAS  Google Scholar 

  15. Sun, J.S. & Manley, J.L. A novel U2–U6 snRNA structure is necessary for mammalian mRNA splicing. Genes Dev. 9, 843–854 (1995).

    Article  CAS  Google Scholar 

  16. Hilliker, A.K. & Staley, J.P. Multiple functions for the invariant AGC triad of U6 snRNA. RNA 10, 921–928 (2004).

    Article  CAS  Google Scholar 

  17. Datta, B. & Weiner, A.M. The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 5377–5382 (1993).

    Article  CAS  Google Scholar 

  18. Field, D.J. & Friesen, J.D. Functionally redundant interactions between U2 and U6 spliceosomal snRNAs. Genes Dev. 10, 489–501 (1996).

    Article  CAS  Google Scholar 

  19. Yan, D. & Ares, M. Jr. Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits. Mol. Cell. Biol. 16, 818–828 (1996).

    Article  CAS  Google Scholar 

  20. Fortner, D.M., Troy, R.G. & Brow, D.A. A stem/loop in U6 RNA defines a conformational switch required for pre-mRNA splicing. Genes Dev. 8, 221–233 (1994).

    Article  CAS  Google Scholar 

  21. Huppler, A., Nikstad, L.J., Allmann, A.M., Brow, D.A. & Butcher, S.E. Metal binding and base ionization in the U6 RNA intramolecular stem-loop structure. Nat. Struct. Biol. 9, 431–435 (2002).

    Article  CAS  Google Scholar 

  22. Yean, S.L., Wuenschell, G., Termini, J. & Lin, R.J. Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome. Nature 408, 881–884 (2000).

    Article  CAS  Google Scholar 

  23. Sigel, R.K., Vaidya, A. & Pyle, A.M. Metal ion binding sites in a group II intron core. Nat. Struct. Biol. 7, 1111–1116 (2000).

    Article  CAS  Google Scholar 

  24. Boulanger, S.C. et al. Studies of point mutants define three essential paired nucleotides in the domain 5 substructure of a group II intron. Mol. Cell. Biol. 15, 4479–4488 (1995).

    Article  CAS  Google Scholar 

  25. Clore, G.M. & Kuszewski, J. Improving the accuracy of NMR structures of RNA by means of conformational database potentials of mean force as assessed by complete dipolar coupling cross-validation. J. Am. Chem. Soc. 125, 1518–1525 (2003).

    Article  CAS  Google Scholar 

  26. Reiter, N.J., Nikstad, L.J., Allmann, A.M., Johnson, R.J. & Butcher, S.E. Structure of the U6 RNA intramolecular stem-loop harboring an S(P)-phosphorothioate modification. RNA 9, 533–542 (2003).

    Article  CAS  Google Scholar 

  27. Peebles, C.L., Zhang, M., Perlman, P.S. & Franzen, J.S. Catalytically critical nucleotide in domain 5 of a group II intron. Proc. Natl. Acad. Sci. USA 92, 4422–4426 (1995).

    Article  CAS  Google Scholar 

  28. Zhang, L. & Doudna, J.A. Structural insights into group II intron catalysis and branch-site selection. Science 295, 2084–2088 (2002).

    Article  CAS  Google Scholar 

  29. Sigel, R.K. et al. Solution structure of domain 5 of a group II intron ribozyme reveals a new RNA motif. Nat. Struct. Mol. Biol. 11, 187–192 (2004).

    Article  CAS  Google Scholar 

  30. Ryan, D.E. & Abelson, J. The conserved central domain of yeast U6 snRNA: importance of U2–U6 helix Ia in spliceosome assembly. RNA 8, 997–1010 (2002).

    Article  CAS  Google Scholar 

  31. Fabrizio, P. & Abelson, J. Point mutations in yeast U6 snRNA can specifically block the first or second step of pre-mRNA splicing in vitro. Mol. Biol. Rep. 14, 135 (1990).

    Article  CAS  Google Scholar 

  32. Hohng, S. et al. Conformational flexibility of four-way junctions in RNA. J. Mol. Biol. 336, 69–79 (2004).

    Article  CAS  Google Scholar 

  33. Rupert, P.B. & Ferre-D'Amare, A.R. Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis. Nature 410, 780–786 (2001).

    Article  CAS  Google Scholar 

  34. Tani, T. & Ohshima, Y. mRNA-type introns in U6 small nuclear RNA genes: implications for the catalysis in pre-mRNA splicing. Genes Dev. 5, 1022–1031 (1991).

    Article  CAS  Google Scholar 

  35. Tarn, W.Y. & Steitz, J.A. Highly diverged U4 and U6 small nuclear RNAs required for splicing rare AT-AC introns. Science 273, 1824–1832 (1996).

    Article  CAS  Google Scholar 

  36. Konforti, B.B. et al. Ribozyme catalysis from the major groove of group II intron domain 5. Mol. Cell 1, 433–441 (1998).

    Article  CAS  Google Scholar 

  37. Madhani, H.D. & Guthrie, C. Randomization-selection analysis of snRNAs in vivo: evidence for a tertiary interaction in the spliceosome. Genes Dev. 8, 1071–1086 (1994).

    Article  CAS  Google Scholar 

  38. Hansen, M.R., Hanson, P. & Pardi, A. Filamentous bacteriophage for aligning RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions. Methods Enzymol. 317, 220–240 (2000).

    Article  CAS  Google Scholar 

  39. Ottiger, M., Delaglio, F., Marquardt, J.L., Tjandra, N. & Bax, A. Measurement of dipolar couplings for methylene and methyl sites in weakly oriented macromolecules and their use in structure determination. J. Magn. Reson. 134, 365–369 (1998).

    Article  CAS  Google Scholar 

  40. Brunger, A.T. et al. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  41. Sashital, D.G., Allmann, A.M., Van Doren, S.R. & Butcher, S.E. Structural basis for a lethal mutation in U6 RNA. Biochemistry 42, 1470–1477 (2003).

    Article  CAS  Google Scholar 

  42. Schwieters, C.D., Kuszewski, J.J., Tjandra, N. & Marius Clore, G. The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank C.J. McManus for sample preparation, and M. Tonelli and the National Magnetic Resonance Facility (NMRFAM) staff for technical support. NMR studies were carried out at NMRFAM with support from the US National Institutes of Health (NIH) Biomedical Technology Program and additional equipment funding from the University of Wisconsin, the US National Science Foundation (NSF) Academic Infrastructure Program, the NIH Shared Instrumentation Program, the NSF Biological Instrumentation Program, and the US Department of Agriculture. This investigation was supported by NIH grant GM65166 to S.E.B. and by NIH predoctoral training grant T32 GM007215 to D.G.S.

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  1. Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, 53706, Wisconsin, USA

    Dipali G Sashital, Gabriel Cornilescu & Samuel E Butcher

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  1. Dipali G Sashital
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  2. Gabriel Cornilescu
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Correspondence to Samuel E Butcher.

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Supplementary information

Supplementary Fig. 1

U2-U6 complexes used in NMR study. (PDF 78 kb)

Supplementary Fig. 2

NMR evidence for four-helix junction in all U2–U6 complexes. (PDF 126 kb)

Supplementary Fig. 3

Overlay of 2D NOESY sequential region for extended U6 ISL and U2–U6 complex. (PDF 123 kb)

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Sashital, D., Cornilescu, G. & Butcher, S. U2–U6 RNA folding reveals a group II intron-like domain and a four-helix junction. Nat Struct Mol Biol 11, 1237–1242 (2004). https://doi.org/10.1038/nsmb863

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