title: Cryo-EM structure of the spliceosome immediately after branching.
abstract: Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5'-splice site is cleaved but remains close to the catalytic Mg2+site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5'-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2'OH. The 5'-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5'-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Wojciech P
authors.initials: WP
authors.lastname: Galej
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Max E
authors.initials: ME
authors.lastname: Wilkinson
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Sebastian M
authors.initials: SM
authors.lastname: Fica
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Chris
authors.initials: C
authors.lastname: Oubridge
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Newman
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Nagai
mesh_terms: D000241:Adenosine / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D020134:Catalytic Domain; D020285:Cryoelectron Microscopy; D004951:Esterification; D005091:Exons / Q000235:genetics; D007438:Introns / Q000235:genetics; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing; D000072260:RNA Splicing Factors / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D017381:Spliceosomes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*
chemical_list: C544745:Cwc25 protein, S cerevisiae; D009687:Nuclear Proteins; C077018:PRP8 protein, S cerevisiae; D012322:RNA Precursors; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405484:U2 small nuclear RNA; C405485:U5 small nuclear RNA; C405486:U6 small nuclear RNA; C075469:YJU2 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; D020365:RNA Helicases; D008274:Magnesium; D000241:Adenosine