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Abstract：
U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer\'s disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of full-length U1-70K from E. coli Support Protocol 1: Making chemically competent BL21 Star pRARE/pBB535 cells Basic Protocol 2: Phosphorylation of full-length U1-70K using SRPK1 Support Protocol 2: Purification of SRPK1 Basic Protocol 3: Expression and purification of U1-70K BAD1 from E. coli Basic Protocol 4: Phosphorylation of U1-70K BAD1 using SRPK1 Basic Protocol 5: Expression and purification of U1-70K BAD2 from E. coli.
摘要：
U1-70K（snRNP70）是U1复合物中不可或缺的蛋白质成分，假设在组成型和选择性RNA剪接过程中起关键作用。值得注意的是,U1-70K参与与SR蛋白的相互作用，煽动剪接体的组装。该蛋白质通过多个位点的磷酸化经历调节。非常感兴趣，U1-70K与阿尔茨海默病有关，其中它倾向于形成洗涤剂不溶性聚集体。尽管它是在三十多年前被发现的，我们对U1-70K的理解仍然受到很大限制，主要是由于挑战，如低水平的重组表达，对蛋白质降解的敏感性，和不溶性。为了解决这些限制，我们设计了一个包含密码子优化的多方面方法，战略净化，和增溶方案。这种方法使我们能够实现全长的高产量，可溶性U1-70K，为其全面的生物物理和生化表征铺平了道路。此外，我们提供了制备磷酸化U1-70K的详细方案。这组协议有望成为科学家在RNA剪接的背景下探索U1-70K相关机制的复杂网络及其在神经退行性疾病和其他疾病和生物过程中的影响的宝贵资源。©2024作者WileyPeriodicalsLLC出版的当前协议。基本方案1:来自大肠杆菌的全长U1-70K的表达和纯化支持方案1:制备化学上合格的BL21StarpRARE/pBB535细胞基本方案2:使用SRPK1磷酸化全长U1-70K支持方案2:SRPK1的纯化基本方案3:来自大肠杆菌的U1-70KBAD1的表达和纯化基本方案4:来自SRcoli的B70K的磷酸化纯化方案E.
