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. But whether splicing is catalysed by small nuclear RNAs in the spliceosome is unresolved. As the spliceosome is a metalloenzyme, 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.
Yean
S L
SL
Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010-3011, USA.
Wuenschell
G
G
Termini
J
J
Lin
R J
RJ
D002384:Catalysis; D004952:Esters / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism; D015003:Yeasts
D004952:Esters; D012322:RNA Precursors; D012331:RNA, Fungal; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D013873:Thionucleotides; C405486:U6 small nuclear RNA; D008345:Manganese; D008274:Magnesium