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<p>Formation of the activated human spliceosome (B<sup>act</sup>) involves major structural rearrangements, leading to the catalytically active U2/U6 RNA core. This process involves at least two intermediates, pre-B<sup>act-1</sup> and pre-B<sup>act-2</sup>, and is regulated by CDK11-mediated phosphorylation of the U2 snRNP protein SF3B1. However, the mechanisms of this essential step are poorly understood. Here we present the cryo-EM structure of a spliceosome stalled – by the CDK11 inhibitor OTS964 – in a previously undescribed early-activated state, termed pre-B<sup>act-OTS</sup>, shortly after dissociation of U4 snRNP. In pre-B<sup>act-OTS</sup>, the U2-SF3B6 protein is retained in a C-terminal region of the super-helical U2-SF3B1 HEAT domain (SF3B1<sup>HEAT</sup>) that clamps the U2/branch-site helix. In contrast, in pre-B<sup>act-1</sup>, SF3B6 is repositioned to SF3B1’s N-terminal HEAT repeats, thereby preventing a steric clash of SF3B6 with PRP8 during the pre-B<sup>act-OTS</sup>-to-pre-B<sup>act-1</sup> transition. We infer that the CDK11-mediated phosphorylation of SF3B1 drives the relocation of SF3B6, gating progression to B<sup>act</sup> formation. In pre-B<sup>act-OTS</sup>, we also located the RNA helicase DHX15 at the N-terminal region of SF3B1<sup>HEAT</sup>, assisted by the SR140/SPF45/CHERP/SUGP1 protein complex. These results suggest the involvement of DHX15 in kinase-mediated proofreading of the early-activated spliceosome, by competing with CDK11’s phosphorylation of SF3B1, and thus with relocation of SF3B6 at SF3B1<sup>HEAT</sup>.</p>

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Structural basis of the regulation by CDK11 kinase of early spliceosome activation and evidence for its proofreading by DHX15 helicase

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月3日

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  • Zhenwei Zhang,
  • Vinay Kumar,
  • Sumeru Panta,
  • Jiayun Zhong,
  • Olexandr Dybkov,
  • Haohao Dong,
  • Berthold Kastner,
  • Henning Urlaub,
  • Holger Stark,
  • Reinhard Lührmann

摘要

Formation of the activated human spliceosome (Bact) involves major structural rearrangements, leading to the catalytically active U2/U6 RNA core. This process involves at least two intermediates, pre-Bact-1 and pre-Bact-2, and is regulated by CDK11-mediated phosphorylation of the U2 snRNP protein SF3B1. However, the mechanisms of this essential step are poorly understood. Here we present the cryo-EM structure of a spliceosome stalled – by the CDK11 inhibitor OTS964 – in a previously undescribed early-activated state, termed pre-Bact-OTS, shortly after dissociation of U4 snRNP. In pre-Bact-OTS, the U2-SF3B6 protein is retained in a C-terminal region of the super-helical U2-SF3B1 HEAT domain (SF3B1HEAT) that clamps the U2/branch-site helix. In contrast, in pre-Bact-1, SF3B6 is repositioned to SF3B1’s N-terminal HEAT repeats, thereby preventing a steric clash of SF3B6 with PRP8 during the pre-Bact-OTS-to-pre-Bact-1 transition. We infer that the CDK11-mediated phosphorylation of SF3B1 drives the relocation of SF3B6, gating progression to Bact formation. In pre-Bact-OTS, we also located the RNA helicase DHX15 at the N-terminal region of SF3B1HEAT, assisted by the SR140/SPF45/CHERP/SUGP1 protein complex. These results suggest the involvement of DHX15 in kinase-mediated proofreading of the early-activated spliceosome, by competing with CDK11’s phosphorylation of SF3B1, and thus with relocation of SF3B6 at SF3B1HEAT.