Abstract：
Single-nucleotide variants (SNVs) in the gene encoding Kinesin Family Member 5A (KIF5A), a neuronal motor protein involved in anterograde transport along microtubules, have been associated with amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive and fatal neurodegenerative disease that primarily affects the motor neurons. Numerous ALS-associated KIF5A SNVs are clustered near the splice-site junctions of the penultimate exon 27 and are predicted to alter the carboxy-terminal (C-term) cargo-binding domain of KIF5A. Mis-splicing of exon 27, resulting in exon exclusion, is proposed to be the mechanism by which these SNVs cause ALS. Whether all SNVs proximal to exon 27 result in exon exclusion is unclear. To address this question, we designed an in vitro minigene splicing assay in human embryonic kidney 293 cells, which revealed heterogeneous site-specific effects on splicing: only 5\' splice-site (5\'ss) SNVs resulted in exon skipping. We also quantified splicing in select clustered, regularly interspaced, short palindromic repeats-edited human stem cells, differentiated to motor neurons, and in neuronal tissues from a 5\'ss SNV knock-in mouse, which showed the same result. Moreover, the survival of representative 3\' splice site, 5\'ss, and truncated C-term variant KIF5A (v-KIF5A) motor neurons was severely reduced compared with wild-type motor neurons, and overt morphological changes were apparent. While the total KIF5A mRNA levels were comparable across the cell lines, the total KIF5A protein levels were decreased for v-KIF5A lines, suggesting an impairment of protein synthesis or stability. Thus, despite the heterogeneous effect on ribonucleic acid splicing, KIF5A SNVs similarly reduce the availability of the KIF5A protein, leading to axonal transport defects and motor neuron pathology.
摘要：
编码驱动蛋白家族成员5A（KIF5A）的基因中的单核苷酸变体（SNV），一种参与微管顺行运输的神经元运动蛋白，与肌萎缩侧索硬化症（ALS）有关。ALS是一种快速进展和致命的神经退行性疾病，主要影响运动神经元。许多ALS相关的KIF5ASNV聚集在倒数第二个外显子27的剪接位点连接处附近，并预测会改变KIF5A的羧基末端（C端）货物结合域。外显子27的错误剪接，导致外显子排斥，被认为是这些SNV引起ALS的机制。是否所有邻近外显子27的SNV导致外显子排除尚不清楚。为了解决这个问题，我们在HEK293细胞中设计了一种体外小基因剪接测定法，该测定法揭示了对剪接的异质位点特异性作用：只有5'剪接位点（5'ss）SNV导致外显子跳跃。我们还量化了分化为运动神经元的选择CRISPR编辑的人干细胞和5'ssSNV敲入小鼠的神经元组织中的剪接，显示出相同的结果。此外，代表性3\'剪接位点的存活，5\'ss，与野生型运动神经元相比，截短的C-term变体KIF5A（v-KIF5A）运动神经元严重减少，明显的形态变化。虽然KIF5AmRNA的总水平在细胞系中具有可比性，v-KIF5A系的总KIF5A蛋白水平降低，提示蛋白质合成或稳定性受损。因此，尽管对RNA剪接有异质作用，KIF5ASNV同样降低了KIF5A蛋白的可用性，导致轴突运输缺陷和运动神经元病理。
