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. 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

前体mRNA（pre-mRNA）剪接过程中的内含子切除步骤极为复杂，其功能异常会导致人类大量遗传性疾病。尽管经典剪接体（canonical spliceosome）循环的关键环节已通过结构-功能联合分析得以阐明，但目前尚无关于注定被清除的异常剪接体的结构信息。本研究解析了预装配为拆卸状态的B态剪接体中间体的冷冻电镜（cryo-EM）结构。我们鉴定到DEAH盒解旋酶-G补丁结构域蛋白对（Gih35-Gpl1）可维持剪接体的催化休眠状态：由于5’外显子3’端存在单核苷酸插入，Gpl1能够识别剪接体经重塑后的活性位点。该重塑信号被传递至剪接体表面，进而招募Ntr1复合物（Ntr1 complex）。本研究的数据为剪接体相关质量控制（spliceosome-associated quality control）的靶向分析奠定了基础。本研究通过高通量poly(A)+ RNA测序对Ctr1缺陷型与野生型（WT）菌株进行了详细分析；同时对携带Nrl1-NTP_Prp43-6xFlag标签的粟酒裂殖酵母（S. pombe）菌株开展了RNA免疫沉淀（RNA-IP）测序实验（含2个生物学重复），并以无标签对照菌株的RNA-IP测序作为阴性对照，此外还完成了总RNA测序。