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

• Published in: Nature (London) Vol. 408; no. 6814; pp. 881 - 884
• Main Authors: Lin, Ren-Jang, Yean, Shyue-Lee, Wuenschell, Gerald, Termini, John
• Format: Journal Article
• Language: English
• Published: London Nature Publishing 14.12.2000; Nature Publishing Group
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Catalysis; Esters - metabolism; Fundamental and applied biological sciences. Psychology; Ions; Magnesium - metabolism; Manganese - metabolism; Metal ions; Metals; Molecular biology; Nucleic acids; Ribonucleic acid; RNA; RNA Precursors - metabolism; RNA Splicing; RNA, Fungal - chemistry; RNA, Fungal - metabolism; RNA, Messenger - metabolism; Rna, ribonucleoproteins; RNA, Small Nuclear - chemistry; RNA, Small Nuclear - metabolism; snRNA U6; spliceosomes; Spliceosomes - metabolism; Thionucleotides - metabolism; Yeast; Yeasts; Spliceosome; Splicing; RNA; Metal ion; Molecular complex; Oligonucleotide; Catalysis; Metalloenzyme; Protein
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/35048617

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.