Structure of an aberrant spliceosome intermediate on its way to disassembly. Structure of an aberrant spliceosome intermediate on its way to disassembly

Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses, structural information on an aberrant spliceosome committed to discard is not available. Here, we report the cryo-EM structure of a B state spliceosome intermediate primed for disassembly. We identify the DEAH-box helicase – G patch protein pair (Gih35-Gpl1) to maintain catalytic dormancy with Gpl1 recognizing a remodeled active site of the spliceosome due to a single-nucleotide insertion at the 3’ end of the 5’ exon. Remodeling is communicated to the spliceosome surface and the Ntr1 complex is recruited. Our data pave the way for a targeted analysis of spliceosome-associated quality control. Overall design: Detailed analysis of Ctr1-deficient and WT strains using high throughput poly(A)+ RNA sequencing. RNAseq of RNA-IP experiemnts of Nrl1-NTP_Prp43-6xFlag S. pombe strains (2 biological repliactes), RNA-IP of no-tag control strain (negative control) and total RNA sequencing

前信使RNA（pre-mRNA）剪接过程中的内含子移除机制极为复杂，其功能紊乱会引发人类大量遗传性疾病。尽管结合结构与功能分析已揭示了经典剪接体循环的关键步骤，但目前尚无关于倾向于被清除的异常剪接体的结构信息。本研究解析了一个准备进行拆解的B态剪接体中间复合物的冷冻电镜（cryo-EM）结构。我们鉴定到DEAH盒解旋酶-G结构域蛋白配对（Gih35-Gpl1）可维持剪接体的催化休眠状态：Gpl1可识别因5’外显子3’端发生单核苷酸插入而重塑的剪接体活性位点。该重塑信号会传递至剪接体表面，并招募Ntr1复合物。本研究数据为剪接体相关质量控制的靶向分析提供了研究基础。实验设计：通过高通量聚腺苷酸化RNA（poly(A)+ RNA）测序技术，对Ctr1缺陷型与野生型（WT）菌株进行详细分析。对Nrl1-NTP_Prp43-6xFlag粟酒裂殖酵母（S. pombe）菌株进行RNA免疫沉淀（RNA-IP）实验的RNA测序（共2次生物学重复）、无标签对照菌株的RNA-IP实验（阴性对照）以及总RNA测序。