Abstract
Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome's catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The 3' region of the U2 snRNP is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5' splice site. The U6 ACAGAGA sequence forms a hairpin that weakly tethers the 5' splice site. The B complex proteins Prp38, Snu23 and Spp381 bind the Prp8 N-terminal domain and stabilize U6 ACAGAGA stem-pre-mRNA and Brr2-U4 small nuclear RNA interactions. These results provide important insights into the events leading to active site formation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Base Sequence
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Biocatalysis
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Catalytic Domain
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Cryoelectron Microscopy*
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Introns / genetics
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Models, Biological
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Models, Molecular
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Nuclear Proteins / chemistry
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Nuclear Proteins / metabolism
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Protein Binding
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Protein Domains
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Protein Stability
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RNA Helicases / chemistry
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RNA Helicases / metabolism
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RNA Helicases / ultrastructure
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RNA Precursors / genetics
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RNA Precursors / metabolism
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RNA Precursors / ultrastructure
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RNA Splice Sites / genetics
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RNA Splicing
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RNA Splicing Factors / chemistry
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RNA Splicing Factors / metabolism
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RNA, Small Nuclear / chemistry
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RNA, Small Nuclear / metabolism
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Ribonucleoprotein, U2 Small Nuclear / chemistry
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Ribonucleoprotein, U2 Small Nuclear / metabolism
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Ribonucleoprotein, U4-U6 Small Nuclear / chemistry
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Ribonucleoprotein, U4-U6 Small Nuclear / metabolism
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Ribonucleoprotein, U5 Small Nuclear / chemistry
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Ribonucleoprotein, U5 Small Nuclear / metabolism
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Ribonucleoproteins, Small Nuclear / chemistry
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Ribonucleoproteins, Small Nuclear / metabolism
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / metabolism
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Saccharomyces cerevisiae Proteins / ultrastructure
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Saccharomyces cerevisiae* / chemistry
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Saccharomyces cerevisiae* / genetics
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Spliceosomes / chemistry*
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Spliceosomes / metabolism
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Spliceosomes / ultrastructure*
Substances
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Nuclear Proteins
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PRP38 protein, S cerevisiae
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PRP8 protein, S cerevisiae
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RNA Precursors
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RNA Splice Sites
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RNA Splicing Factors
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RNA, Small Nuclear
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Ribonucleoprotein, U2 Small Nuclear
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Ribonucleoprotein, U4-U6 Small Nuclear
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Ribonucleoprotein, U5 Small Nuclear
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Ribonucleoproteins, Small Nuclear
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Saccharomyces cerevisiae Proteins
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Snu23 protein, S cerevisiae
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Spp381 protein, S cerevisiae
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U6 small nuclear RNA
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BRR2 protein, S cerevisiae
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RNA Helicases