Abstract
Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA–protein complex called the spliceosome1,2. But whether splicing is catalysed by small nuclear RNAs3,4 in the spliceosome is unresolved. As the spliceosome is a metalloenzyme5,6,7, it is important to determine whether snRNAs coordinate catalytic metals. Here we show that yeast U6 snRNA coordinates a metal ion that is required for the catalytic activity of the spliceosome. With Mg2+, U6 snRNA with a sulphur substitution for the pro-RP or pro-SP non-bridging phosphoryl oxygen of nucleotide U80 reconstitutes a fully assembled yet catalytically inactive spliceosome. Adding a thiophilic ion such as Mn2+ allows the first transesterification reaction to occur in the U6/sU80(SP)- but not the U6/sU80(RP)-reconstituted spliceosome. Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the SP spliceosome. Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.
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Acknowledgements
We thank J. Abelson for U6 plasmids; D. McPheeters and D. Ryan for advice on RNA ligation procedure; and T. Nilsen, E. Sontheimer, D. McPheeters, J. Rossi, G. Edwalds-Gilbert and E. Silverman for critical reading of the manuscript. This work is supported by grants from NIH to J.T. and R.-J.L. The Molecular Dynamics phosphorimager was purchased with a core grant from NSF.
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Yean, SL., Wuenschell, G., Termini, J. et al. Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome. Nature 408, 881–884 (2000). https://doi.org/10.1038/35048617
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DOI: https://doi.org/10.1038/35048617
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