psiCLIP 揭示了 DEAH -box 解旋酶在exon 连接前后的动态 RNA 结合。
psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation.
机构信息
MRC Laboratory of Molecular Biology, Cambridge, UK.
The Francis Crick Institute, London, UK.
出版信息
Nat Commun. 2021 Mar 5;12(1):1488. doi: 10.1038/s41467-021-21745-9.
RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3'-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.
RNA 解旋酶重塑剪接体以促进前体 mRNA 的剪接，但它们的结合和作用机制仍知之甚少。为了确定特定剪接体状态下解旋酶-RNA 的接触，我们开发了纯化的剪接体 iCLIP（psiCLIP），它揭示了剪接催化过程中动态解旋酶-RNA 的接触。解旋酶 Prp16 沿着分支点和 3'-剪接位点之间的整个可用单链 RNA 区域结合，而 Prp22 在 exon 连接之前在分支点下游弥散结合，但在 exon 连接后在下游 exon 中切换到更窄的结合，这表明不是连续易位的机制。exon 连接因子 Prp18 的耗竭会破坏 Prp22 与前体 mRNA 的结合，这表明 Prp22 的校对可能在 exon 连接过程中感知剪接体的稳定性。因此，psiCLIP 通过提供对剪接体 RNA 解旋酶结合和校对活性的关键见解，补充了结构研究。
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