Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [RNA-Seq]. Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles [RNA-Seq]

A major impediment to studying the human spliceosome in vivo has been the inability to program point mutations in endogenous genes on a large scale. CRISPR-Cas9 technology now provides such opportunities. Due to its scalability and ability to introduce point mutations, we chose CRISPR-Cas9 base editing for a forward genetic screen of the spliceosome in an eHAP haploid human cell line background. We maintained eHAP FNLS cells as haploid so that we could subsequently assign genotype-phenotype relationships. We designed a single guide RNA (sgRNA) library targeting a hand-curated list of 153 human spliceosomal proteins which engage at various steps of the splicing cycle. After mutagenesis, we interrogated the spliceosome using the potent inhibitor pladienolide B (PB), which targets U2 snRNP by binding to a pocket between the SF3B1 and PHF5A subunits of the SF3b complex, preventing stabilization of the U2-branchpoint RNA duplex. Validation and genomic sequencing revealed resistance mutations in SF3B1 and PHF5A in residues adjacent to the compound binding pocket. We mapped hypersensitive mutants to U2 snRNP components, but also to factors that act as late as the second chemical step, after SF3b has dissociated. Strikingly, we obtained resistance mutants in SUGP1, a spliceosomal G-patch protein of unknown function that lacks orthologs in yeast and is also a newly proposed tumor suppressor whose loss underpins the splicing changes induced by cancer-associated SF3B1 mutations. Overall design: RNA-seq to investigate the effect of identified mutations in the spliceosomal proteins PHF5A, SF3B1 and SUGP1 (linked to resistance to the small molecule spliceosome inhibitor pladienolide B) on pre-mRNA splicing.

体内研究人类剪接体（spliceosome）的主要障碍，在于无法大规模对内源基因（endogenous genes）进行点突变（point mutations）编程。如今CRISPR-Cas9技术为该问题提供了可行解决方案。鉴于其可扩展性与引入点突变的能力，我们选择CRISPR-Cas9碱基编辑（base editing）技术，在eHAP单倍体人类细胞系（eHAP haploid human cell line）背景下开展剪接体的正向遗传筛选（forward genetic screen）。为后续能够建立基因型-表型关联（genotype-phenotype relationships），我们维持eHAP FNLS细胞的单倍体状态。我们设计了靶向经人工精选（hand-curated）的153个人类剪接体蛋白的单向导RNA（single guide RNA, sgRNA）文库，这些蛋白参与剪接周期（splicing cycle）的不同步骤。诱变处理（mutagenesis）后，我们利用强效抑制剂普拉迪诺利德B（pladienolide B, PB）探究剪接体功能：PB通过结合SF3b复合物（SF3b complex）中SF3B1与PHF5A亚基之间的结合口袋，靶向U2小核糖核蛋白颗粒（U2 snRNP），阻止U2-分支点RNA双链体（U2-branchpoint RNA duplex）的稳定。验证实验与基因组测序（genomic sequencing）结果显示，SF3B1与PHF5A中与化合物结合口袋相邻的残基存在耐药突变（resistance mutations）。我们将超敏突变体（hypersensitive mutants）定位至U2 snRNP组分，同时也定位至SF3b解离后才发挥作用的晚期剪接因子。值得注意的是，我们在SUGP1中获得了耐药突变：SUGP1是一种功能未知的剪接体G-补丁结构域蛋白（G-patch protein），在酵母中无直系同源物（orthologs），同时也是新提出的肿瘤抑制因子（tumor suppressor），其缺失会介导癌症相关SF3B1突变诱导的前体mRNA剪接（pre-mRNA splicing）改变。总体实验设计：通过RNA测序，探究已鉴定的剪接体蛋白PHF5A、SF3B1与SUGP1（与小分子剪接体抑制剂普拉迪诺利德B耐药相关）的突变对前体mRNA剪接的影响。