Structural insights into the cross-exon to cross-intron spliceosome switch

• Published in: Nature (London) 2024-06, Vol.630 (8018), p.1012-1019
• Main Authors: Zhang, Zhenwei, Kumar, Vinay, Dybkov, Olexandr, Will, Cindy L., Zhong, Jiayun, Ludwig, Sebastian E. J., Urlaub, Henning, Kastner, Berthold, Stark, Holger, Lührmann, Reinhard
• Format: Article
• Language: English
• Subjects: 101/28; 101/58; 631/337/1645/1792; 631/535/1258/1259; Alternative Splicing; Assembly; Catalysis; Cryoelectron Microscopy; Electron microscopes; Electron microscopy; Exons - genetics; Humanities and Social Sciences; Humans; Introns - genetics; Microscopy; Models, Molecular; Molecular modelling; mRNA; multidisciplinary; Proteins; Ribonucleoproteins (small nuclear); Ribonucleoproteins, Small Nuclear - chemistry; Ribonucleoproteins, Small Nuclear - metabolism; Ribonucleoproteins, Small Nuclear - ultrastructure; RNA Splice Sites - genetics; RNA Splicing - genetics; Science; Science (multidisciplinary); Spliceosomes - chemistry; Spliceosomes - metabolism; Spliceosomes - ultrastructure; Transmission electron microscopy; Upstream
• ISSN: 0028-0836; 1476-4687; 1476-4687
• DOI: 10.1038/s41586-024-07458-1

Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron 1 . Alternatively, it can occur through an exon-defined pathway 2 – 5 , whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5′ splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex 6 , 7 . Exon definition promotes the splicing of upstream introns 2 , 8 , 9 and plays a key part in alternative splicing regulation 10 – 16 . However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5′ splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage. Cryo-electron microscopy structures of cross-exon pre-B and B-like complexes contribute new insights into the molecular mechanisms that mediate the switch from a cross-exon to a cross-intron organized spliceosome.