Two oppositely-chargedsf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila

Somatic SF3b1 mutations occur in many cancers, and the highly conserved H662 residue is one of the most frequently mutated sites. To address their mechanism on splicing alteration and effects on development, we constructed mutant strains at the corresponding residue H698 in Drosophila using CRISPR-Cas9 system. Two mutations, H698D and H698R, were selected due to their presence in patients and the significantly opposite charges. Both the sf3b1-H698D and –H698R mutant flies exhibit developmental defects including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespan. Interestingly, the H698D mutant has decreased resistance to fungi infection, while the H698R mutant has impaired climbing ability. Further RNA-seq data finds altered expression of immunity response genes and changed alternative splicing of muscle and neural-related genes in the two mutants respectively. Lariat sequencing reveal that the sf3b1-H662 mutations cause aberrant selection of multiple intronic branch sites and the H698R mutant prefers to use upstream branch sites in the alternative splicing changed events. This study provides in vivo evidence from Drosophila to elucidate how the highly conserved SF3b1 cancer mutations alter splicing and their consequences in the development and immune systems. Overall design: Transcriptional profiles of the 5d adults WT, SF3b1-H698D and -H698R mutant strains

体细胞SF3B1（SF3b1）突变在多种癌症中均有发生，其高度保守的H662残基是最常见的突变位点之一。为阐明此类突变介导剪接异常的分子机制及其对生物体发育的影响，本研究利用CRISPR-Cas9基因编辑系统，在果蝇中对应于人类H662的H698残基位点构建了突变株系。本研究选取了H698D与H698R两种突变体，二者均在癌症患者中被检出，且所带电荷存在显著差异。sf3b1-H698D与sf3b1-H698R突变果蝇均表现出发育缺陷表型，包括产卵量减少、孵化率降低、形态发生延迟以及寿命缩短。值得注意的是，H698D突变体对真菌侵染的抗性显著下降，而H698R突变体则出现攀爬能力受损的表型。后续RNA测序（RNA-seq）数据显示，两种突变体中免疫应答基因的表达谱发生显著改变，且肌肉与神经相关基因的可变剪接事件分别出现异常调控。套索测序（Lariat sequencing）结果表明，sf3b1-H662突变会导致多个内含子分支位点的选择出现异常；而在发生可变剪接异常的事件中，H698R突变体更倾向于使用上游分支位点。本研究通过果蝇体内模型提供了直接实验证据，阐明了高度保守的SF3B1癌症突变如何调控剪接过程，及其对发育与免疫系统造成的功能性影响。实验整体设计：对5日龄野生型（WT）、sf3b1-H698D及sf3b1-H698R突变果蝇成虫的转录组谱进行分析。