Use of a novel Förster resonance energy transfer method to identify locations of site-bound metal ions in the U2–U6 snRNA complex

U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its folding and catalytic activity. We used a novel Förster resonance energy transfer (FRET) method between site-bound luminescent lanthanide ions and a covalently attached fluorescent dye, combined with supporting stoichiometric and mutational studies, to determine locations of site-bound Tb(III) within the human U2–U6 complex. At pH 7.2, we detected three metal-ion-binding sites in: (1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; (2) the four-way junction, which contains the conserved AGC triad; and (3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which may impact on splicing activity.

[1]  Kearns Dr,et al.  Europium as a fluorescent probe of transfer RNA structure. , 1975 .

[2]  Europium as a fluorescent probe of transfer RNA structure. , 1975, Biochemistry.

[3]  C. Kittrell,et al.  Laser-induced lanthanide ion laminescence lifetime measurements by direct excitation of metal ion levels. A new class of structural probe for calcium-binding proteins and nucleic acids. , 1977, Journal of the American Chemical Society.

[4]  A Klug,et al.  A crystallographic study of metal-binding to yeast phenylalanine transfer RNA. , 1977, Journal of molecular biology.

[5]  L. Stryer,et al.  Fluorescence energy transfer in the rapid-diffusion limit. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[6]  W. Horrocks,et al.  Lanthanide ion probes of structure in biology. Environmentally sensitive fine structure in laser-induced terbium(III) luminescence. , 1979, Biochimica et biophysica acta.

[7]  W. Horrocks,et al.  Lanthanide ion probes of structure in biology. Laser-induced luminescence decay constants provide a direct measure of the number of metal-coordinated water molecules , 1979 .

[8]  Lanthanide ion probes of structure in biology. Laser-induced luminescence decay constants provide a direct measure of the number of metal-coordinated water molecules , 1979 .

[9]  C. Y. Huang.  Determination of binding stoichiometry by the continuous variation method: the Job plot. , 1982, Methods in enzymology.

[10]  D. Draper.  On the coordination properties of Eu3+ bound to tRNA. , 1985, Biophysical chemistry.

[11]  W. Krzyzosiak,et al.  Identification of the magnesium, europium and lead binding sites in E. coli and lupine tRNAPhe by specific metal ion‐induced cleavages , 1989, FEBS letters.

[12]  C. Evans.  Biochemistry of the Lanthanides , 1990, Biochemistry of the Elements.

[13]  A. Weiner,et al.  Genetic evidence for base pairing between U2 and U6 snRNA in mammalian mRNA splicing , 1991, Nature.

[14]  J. Manley,et al.  Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA , 1991, Nature.

[15]  R. Clegg.  Fluorescence resonance energy transfer and nucleic acids. , 1992, Methods in enzymology.

[16]  Hiten D. Madhani,et al.  A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome , 1992, Cell.

[17]  P. Fabrizio,et al.  Thiophosphates in yeast U6 snRNA specifically affect pre-mRNA splicing in vitro. , 1992, Nucleic acids research.

[18]  J E Hearst,et al.  Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[19]  H. Madhani,et al.  Randomization-selection analysis of snRNAs in vivo: evidence for a tertiary interaction in the spliceosome. , 1994, Genes & development.

[20]  J. Manley,et al.  A novel U2-U6 snRNA structure is necessary for mammalian mRNA splicing. , 1995, Genes & development.

[21]  T. Nilsen,et al.  U6 snRNA function in nuclear pre-mRNA splicing: a phosphorothioate interference analysis of the U6 phosphate backbone. , 1995, RNA.

[22]  W. Mcallister,et al.  Rapid mutagenesis and purification of phage RNA polymerases. , 1997, Protein expression and purification.

[23]  M. Komiyama,et al.  Enormously fast RNA hydrolysis by lanthanide(III) ions under physiological conditions: eminent candidates for novel tools of biotechnology. , 1997, Journal of biochemistry.

[24]  P. Selvin,et al.  Luminescence resonance energy transfer measurements in myosin. , 1998, Biophysical journal.

[25]  O. Uhlenbeck,et al.  Inhibition of the hammerhead ribozyme cleavage reaction by site-specific binding of Tb. , 1997, Science.

[26]  D. Draper,et al.  On the role of magnesium ions in RNA stability , 1998, Biopolymers.

[27]  O. Uhlenbeck,et al.  Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme using luminescence spectroscopy. , 1999, Chemistry & biology.

[28]  D. Turner,et al.  Nuclear magnetic resonance spectroscopy and molecular modeling reveal that different hydrogen bonding patterns are possible for G.U pairs: one hydrogen bond for each G.U pair in r(GGCGUGCC)(2) and two for each G.U pair in r(GAGUGCUC)(2). , 2000, Biochemistry.

[29]  J. Piccirilli,et al.  Metal ion catalysis during the exon-ligation step of nuclear pre-mRNA splicing: extending the parallels between the spliceosome and group II introns. , 2000, RNA.

[30]  D. Lilley,et al.  Location of cyanine-3 on double-stranded DNA: importance for fluorescence resonance energy transfer studies. , 2000, Biochemistry.

[31]  K. Musier-Forsyth,et al.  Use of terbium as a probe of tRNA tertiary structure and folding. , 2000, RNA.

[32]  J. Manley,et al.  A tertiary interaction detected in a human U2-U6 snRNA complex assembled in vitro resembles a genetically proven interaction in yeast. , 2000, RNA.

[33]  P. Selvin,et al.  Quantum yields of luminescent lanthanide chelates and far-red dyes measured by resonance energy transfer. , 2001, Journal of the American Chemical Society.

[34]  S. Butcher.  Structure and function of the small ribozymes. , 2001, Current opinion in structural biology.

[35]  J. Manley,et al.  Splicing-related catalysis by protein-free snRNAs , 2001, Nature.

[36]  N. L. Greenbaum,et al.  Probing of metal-binding domains of RNA hairpin loops by laser-induced lanthanide(III) luminescence. , 2001, Biochemistry.

[37]  D. Brow,et al.  Allosteric cascade of spliceosome activation. , 2002, Annual review of genetics.

[38]  M. Fedor.  The role of metal ions in RNA catalysis. , 2002, Current opinion in structural biology.

[39]  N. L. Greenbaum,et al.  Sequestering of Eu(III) by a GAAA RNA tetraloop. , 2002, Journal of the American Chemical Society.

[40]  Paul R Selvin,et al.  Principles and biophysical applications of lanthanide-based probes. , 2002, Annual review of biophysics and biomolecular structure.

[41]  Taekjip Ha,et al.  Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase , 2002, Nature.

[42]  S. Butcher,et al.  Metal binding and base ionization in the U6 RNA intramolecular stem-loop structure , 2002, Nature Structural Biology.

[43]  Nancy L. Greenbaum,et al.  Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine , 2002, Nature Structural Biology.

[44]  E. Westhof,et al.  The Mg2+ binding sites of the 5S rRNA loop E motif as investigated by molecular dynamics simulations. , 2003, Chemistry & biology.

[45]  J. Manley,et al.  Characterization of the catalytic activity of U2 and U6 snRNAs. , 2003, RNA.

[46]  E. Westhof,et al.  The Mg 2 Binding Sites of the 5 S rRNA Loop E Motif as , 2003 .

[47]  Dipali G. Sashital,et al.  U2–U6 RNA folding reveals a group II intron-like domain and a four-helix junction , 2004, Nature Structural &Molecular Biology.

[48]  Marcia O. Fenley,et al.  Recognition of the spliceosomal branch site RNA helix on the basis of surface and electrostatic features , 2005, Nucleic acids research.

[49]  M. da Graça Miguel,et al.  Changes in hydration of lanthanide ions on binding to DNA in aqueous solution. , 2005, Langmuir : the ACS journal of surfaces and colloids.

[50]  S. Butcher,et al.  Dynamics and metal ion binding in the U6 RNA intramolecular stem-loop as analyzed by NMR. , 2005, Journal of molecular biology.

[51]  Richard A. Stein,et al.  Molecular Imaging: FRET Microscopy and Spectroscopy, A. Periasamy, R.N. Day (Eds.). Oxford University Press Inc. (2005), Price GB £58.00, ISBN: 0-19-517720-6 , 2006 .