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

Abstract

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 RNAs^3,4 in the spliceosome is unresolved. As the spliceosome is a metalloenzyme^5,6,7, 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-R_P or pro-S_P 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(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/U₈₀ 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.