SMN deficiency in spinal muscular atrophy causes widespread intron retention and DNA damage. SMN deficiency in spinal muscular atrophy causes widespread intron retention and DNA damage

Spinal muscular atrophy is the leading genetic cause of infant mortality and is caused by homozygous loss of the SMN1 gene. We investigated global transcriptome changes in the spinal cord of inducible SMA mice. SMN depletion caused widespread retention of introns with weak splice sites or belonging to the minor (U12) class. We further demonstrated accumulation of DNA double strand breaks in the spinal cord of SMA mice and in human SMA cell culture models. DNA damage was partially rescued by suppressing the formation of R-loops, which accumulated over retained introns. We propose that instead of single gene effects, pervasive splicing defects caused by SMN deficiency trigger a global DNA damage and stress response, thus compromising motor neuron survival. Overall design: mRNA-seq: Total spinal cord from SMA and Control mice (rescue experiment) at d20 and d30; human SH-SY5Y cells expressing SMN or Control shRNA for 7d; human iPSC-derived motor neurons expressing SMN or Control shRNA for 5d; total spinal cord from SMA and Control mice (induction experiment) at d10, d20, and d30. DRIP-seq: SH-SY5Y cells expressing SMN or Control shRNA for 7d and immunoprecipitated with S9.6 antibody targeting RNA:DNA hybrids.

脊髓性肌萎缩症（Spinal muscular atrophy, SMA）是引发婴儿死亡的首要遗传性病因，其致病机制为SMN1基因的纯合缺失。本研究对诱导型SMA模型小鼠的脊髓组织开展了全局转录组变化分析，结果显示运动神经元生存（SMN）蛋白缺失会广泛引发内含子滞留，此类滞留内含子多具备较弱的剪接位点，或属于次要剪接体（U12）类内含子。本研究进一步证实，在SMA模型小鼠的脊髓组织以及人类SMA细胞培养模型中，均存在DNA双链断裂的蓄积；而通过抑制在滞留内含子区域蓄积的R环（R-loops）的形成，可部分挽救该DNA损伤。我们提出，SMN蛋白缺失所引发的广泛性剪接缺陷，而非单一基因的异常效应，会触发全局性DNA损伤与应激反应，进而损害运动神经元的存活。
整体实验设计：
mRNA测序（mRNA-seq）：分别采集诱导型SMA模型小鼠与对照小鼠在第20天（d20）、第30天（d30）的全脊髓组织（rescue实验）；将表达SMN靶向短发夹RNA（short hairpin RNA, shRNA）或对照shRNA的人源SH-SY5Y细胞培养7天；将诱导多能干细胞（induced pluripotent stem cell, iPSC）分化得到的人源运动神经元，转染SMN靶向shRNA或对照shRNA并培养5天；另采集诱导型SMA模型小鼠与对照小鼠在第10天（d10）、第20天（d20）、第30天（d30）的全脊髓组织（诱导实验）。
DNA-RNA免疫沉淀测序（DRIP-seq）：将表达SMN靶向shRNA或对照shRNA的人源SH-SY5Y细胞培养7天，使用靶向RNA:DNA杂交链的S9.6抗体进行免疫沉淀。