Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome

Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA-protein complex called the spliceosome(1,2). But whether splicing is catalysed by small nuclear RNAs3,4 in the spliceosome is unresolved. As the spliceosome is a metalloenzyme(5-7), 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-R-P or pro-S-P non-bridging phosphoryl oxygen of nucleotide U-80 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/sU(80)(S-P)- but not the U6/sU80 (R-P)-reconstituted spliceosome. Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U-80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the S-P spliceosome. Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.