U6 snRNA expression prevents toxicity in TDP-43-knockdown cells

Depletion of amyotrophic lateral sclerosis (ALS)-associated transactivation response (TAR) RNA/DNA-binding protein 43 kDa (TDP-43) alters splicing efficiency of multiple transcripts and results in neuronal cell death. TDP-43 depletion can also disturb expression levels of small nuclear RNAs (snRNAs) as spliceosomal components. Despite this knowledge, the relationship between cell death and alteration of snRNA expression during TDP-43 depletion remains unclear. Here, we knocked down TDP-43 in murine neuroblastoma Neuro2A cells and found a time lag between efficient TDP-43 depletion and appearance of cell death, suggesting that several mechanisms mediate between these two events. The amount of U6 snRNA was significantly decreased during TDP-43 depletion prior to increase of cell death, whereas that of U1, U2, and U4 snRNAs was not. Downregulation of U6 snRNA led to cell death, whereas transient exogenous expression of U6 snRNA counteracted the effect of TDP-43 knockdown on cell death, and slightly decreased the mis-splicing rate of Dnajc5 and Sortilin 1 transcripts, which are assisted by TDP-43. These results suggest that regulation of the U6 snRNA expression level by TDP-43 is a key factor in the increase in cell death upon TDP-43 loss-of-function.

肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）相关的反式激活应答（transactivation response, TAR）RNA/DNA结合蛋白43kDa（TDP-43）的耗竭，会改变多种转录本的剪接效率并引发神经元细胞死亡。TDP-43耗竭还会扰乱作为剪接体组分的小核RNA（small nuclear RNAs, snRNAs）的表达水平。尽管已有此类认知，但TDP-43耗竭过程中细胞死亡与snRNA表达改变之间的关联仍不明确。本研究在小鼠神经母细胞瘤Neuro2A细胞中敲低TDP-43，观察到高效TDP-43敲低与细胞死亡出现之间存在时间滞后现象，提示二者之间存在多种介导机制。在细胞死亡水平上升之前的TDP-43耗竭阶段，U6 snRNA的表达量显著降低，而U1、U2及U4 snRNA的表达量无明显变化。下调U6 snRNA会引发细胞死亡，而瞬时外源过表达U6 snRNA则可抵消TDP-43敲低导致的细胞死亡效应，并轻度降低由TDP-43辅助调控的Dnajc5与Sortilin 1转录本的错误剪接率。上述结果表明，TDP-43对U6 snRNA表达水平的调控，是TDP-43功能丧失引发细胞死亡增加的关键因素。