Aberrant alternative splicing plays critical role in aging and age-related diseases. Heterogeneous nuclear ribonucleoproteins (hnRNPs) reportedly regulate RNA splicing process. Whether and how hnRNPs contribute to age-related neurodegenerative diseases, especially Alzheimer’s disease (AD), remain elusive. Immunoblotting and immunostaining were performed to determine expression patterns and cellular/subcellular localization of the long isoform of hnRNP D-like (L-DL), which is a hnRNP family member, in mouse hippocampus. Downregulation of L-DL in WT mice was achieved by AAV-mediated shRNA delivery, followed by memory-related behavioural tests. L-DL interactome was analysed by affinity-precipitation and mass spectrometry. Alternative RNA splicing was measured by RNA-seq and analyzed by bioinformatics-based approaches. Downregulation and upregulation of L-DL in APP/PS1 mice were performed using AAV-mediated transduction. We show that L-DL is specifically localized to nuclear speckles. L-DL levels are decreased in the hippocampus of aged mouse brains and downregulation of L-DL impairs cognition in mice. L-DL serves as a structural component to recruit other speckle proteins, and regulates cytoskeleton- and synapse-related gene expression by altering RNA splicing. Mechanistically, these splicing changes are modulated via L-DL-mediated interaction of SF3B3, a core component of U2 snRNP, and U2AF65, a U2 spliceosome protein that guides U2 snRNP’s binding to RNA. In addition, L-DL levels are decreased in APP/PS1 mouse brains. While downregulation of L-DL deteriorates memory deficits and overexpression of L-DL improves cognitive function in AD mice, by regulating the alternative splicing and expression of synaptic gene CAMKV. Our findings define a molecular mechanism by which hnRNP L-DL regulates alternative RNA splicing, and establish a direct role for L-DL in AD-related synaptic dysfunction and memory decline.

中文翻译：

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核斑点特异性 hnRNP D-like 通过调节 RNA 剪接来防止与年龄和 AD 相关的认知衰退

异常选择性剪接在衰老和与年龄相关的疾病中起着至关重要的作用。据报道，异质核核糖核蛋白 (hnRNPs) 调节 RNA 剪接过程。hnRNPs 是否以及如何导致与年龄相关的神经退行性疾病，尤其是阿尔茨海默病 (AD)，仍然难以捉摸。进行免疫印迹和免疫染色以确定小鼠海马中 hnRNP D 样 (L-DL) 的长同种型的表达模式和细胞/亚细胞定位，它是 hnRNP 家族成员。WT 小鼠中 L-DL 的下调是通过 AAV 介导的 shRNA 递送实现的，然后是与记忆相关的行为测试。通过亲和沉淀和质谱分析 L-DL 相互作用组。通过 RNA-seq 测量替代 RNA 剪接，并通过基于生物信息学的方法进行分析。APP/PS1 小鼠中 L-DL 的下调和上调是使用 AAV 介导的转导进行的。我们表明 L-DL 专门定位于核斑点。老年小鼠大脑海马中的 L-DL 水平降低，L-DL 的下调会损害小鼠的认知能力。L-DL 作为招募其他斑点蛋白的结构成分，并通过改变 RNA 剪接来调节细胞骨架和突触相关基因的表达。从机制上讲，这些剪接变化是通过 L-DL 介导的 SF3B3（U2 snRNP 的核心成分）和 U2AF65（一种引导 U2 snRNP 与 RNA 结合的 U2 剪接体蛋白）的相互作用来调节的。此外，APP/PS1 小鼠大脑中的 L-DL 水平降低。虽然 L-DL 的下调会恶化记忆缺陷，而 L-DL 的过表达会改善 AD 小鼠的认知功能，通过调节突触基因CAMKV的可变剪接和表达。我们的研究结果定义了 hnRNP L-DL 调节选择性 RNA 剪接的分子机制，并确定 L-DL 在 AD 相关突触功能障碍和记忆衰退中的直接作用。