The architecture of the spliceosomal U4/U6.U5 tri-snRNP

U4/U6.U5 tri-snRNP is a 1.5 MDa pre-assembled spliceosomal complex comprising U5 snRNA, extensively base-paired U4/U6 snRNAs and>30 proteins, including the key components Prp8, Brr2 and Snu114. The tri-snRNP combines with a pre-mRNA substrate bound to U1 and U2 snRNPs and transforms into a catalytically active spliceosome following extensive compositional and conformational changes triggered by unwinding of the U4/U6 snRNAs. CryoEM single-particle reconstruction of yeast tri-snRNP at 5.9Å resolution reveals the essentially complete organization of its RNA and protein components. The single-stranded region of U4 snRNA between its 3′-stem-loop and the U4/U6 snRNA stem I is loaded into the Brr2 helicase active site ready for unwinding. Snu114 and the N-terminal domain of Prp8 position U5 snRNA to insert its Loop I, which aligns the exons for splicing, into the Prp8 active site cavity. The structure provides crucial insights into the activation process and the active site of the spliceosome. The protein coding sequences of most eukaryotic genes are interrupted by non-coding segments called introns. Introns are removed from pre-mRNAs and the flanking coding segments (exons) are spliced together to form mRNAs by two successive trans-esterification reactions within a dynamic multi-megadalton protein-RNA complex known as the spliceosome. This complex comprises five canonical subunits, namely U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) and numerous non-snRNP factors1. Each snRNP contains an snRNA, 7 Sm or LSm proteins and a number of snRNP-specific proteins. During the initial stages of spliceosome assembly, U1 and U2 snRNPs recognize Reprints and permissions information is available at www.nature.com/reprints. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http:// www.nature.com/authors/editorial_policies/license.html#terms Correspondence and requests for materials should be addressed to T.H.D.N