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

• 成果类型: Article
• 署名作者: Zhang, Zhenwei; Kumar, Vinay; Dybkov, Olexandr; Will, Cindy L.; Zhong, Jiayun; Ludwig, Sebastian E. J.; Urlaub, Henning; Kastner, Berthold; Stark, Holger; Luehrmann, Reinhard
• 署名单位: Sichuan University; Sichuan University; University of Gottingen; UNIVERSITY GOTTINGEN HOSPITAL
• 刊物名称: Nature
• ISSN/ISSBN: 0028-5474
• DOI: 10.1038/s41586-024-07458-1
• 发表日期: 2024-06-27
• 页码: 1012-+
• 关键词: cryo-em structure; tri-snrnp; u1 snrna; rna; site; definition; protein; atp; RECOGNITION; mechanisms

摘要

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.