Aberrant splicing in Huntingtons disease accompanies disrupted TDP-43 activity and altered m6A RNA modifications [RASL-seq]

Huntingtons disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the HTT gene, leading to altered gene expression. However, the mechanisms leading to disrupted RNA processing in HD remain unclear. Here, we identify the RNA-binding TDP-43 and the N6-methyladenosine (m6A) writer protein METTL3 to be upstream regulators of exon skipping in multiple HD systems. Dysregulated nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 is shown to be present in HD mice and human brains with TDP-43 co-localizing with HTT nuclear aggregate-like bodies distinct from mutant HTT inclusions and from previously observed TDP-43 pathologies. The binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in the striatum from HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a mechanism underlying alternative splicing in HD and highlights the critical nature of TDP-43 loss of function across multiple neurodegenerative diseases. To understand how alternative splicing is altered in HD mouse models, we took the R6/2, Q150, and Q175 HD mouse models and performed RASL-seq with primers spanning short (skipped) and long (not skipped) junctions across splicing sites. We compared the respective mouse to their Wildtype controls. for the Q150 and Q175 we used both heterozygous and homozygous for our experiment.亨廷顿病（Huntingtons disease, HD）是一种由HTT基因第一外显子CAG重复扩增引发的神经退行性疾病，可导致基因表达异常。然而，HD中RNA加工紊乱的具体机制仍未阐明。本研究鉴定出RNA结合蛋白TDP-43（RNA-binding TDP-43）与N6-甲基腺嘌呤（N6-methyladenosine, m6A）写入酶METTL3，为多种HD模型中外显子跳跃事件的上游调控因子。研究证实，HD模型小鼠及人类大脑中存在TDP-43核定位异常与磷酸化TDP-43的胞质蓄积，且TDP-43与HTT核聚集样小体共定位，该结构区别于突变HTT包涵体及既往报道的TDP-43病理改变。同时，编码HD相关差异表达及异常剪接基因的RNA上，TDP-43的结合水平显著降低。最后，HD R6/2模型小鼠纹状体中异常表达的RNA上，m6A修饰水平出现下降，其中包括TDP-43结合位点邻近的成簇修饰位点。本研究结果支持TDP-43功能丧失伴随m6A修饰异常，作为HD中可变剪接紊乱的潜在机制，并凸显了TDP-43功能丧失在多种神经退行性疾病中的关键作用。为明确HD模型小鼠中可变剪接的改变模式，本研究选取R6/2、Q150及Q175三种HD模型小鼠，采用覆盖剪接位点处短（外显子跳跃型）与长（非外显子跳跃型）连接区的引物开展RASL-seq实验，并将各模型小鼠与其对应的野生型对照进行比较。针对Q150与Q175模型，本研究同时使用了杂合子与纯合子样本开展实验。