Analysis of alternative exon-dependent interactome remodelling reveals multitasking functions of gene regulatory factors (Sap30bp RNA-Seq)

Alternative splicing is fundamental for the expansion of biological complexity, particularly in the vertebrate nervous system. Increasing evidence indicates that developmental stage and tissue-dependent alternative exons often control protein-protein interactions, yet only a minor fraction of these events has been characterized at the protein level. Using affinity purification-mass spectrometry (AP-MS) we show that approximately 60% of analyzed neural-differential exons in proteins previously implicated in transcriptional regulation result in the gain or loss of interaction partners. Focusing on Chtop and Sap30bp, the neural exons in these proteins unexpectedly remodel interactions with partners associated with mRNA processing and trafficking. Sap30bp additionally controls RNA localization by regulating the splicing of <100 nt 'mini-introns' that contribute to the nuclear retention of transcripts. These activities of Chtop and Sap30bp are linked to programs of transcriptomic regulation associated with development of the nervous system. AP-MS is thus a powerful approach for elucidating multifaceted functions of proteins imparted by context-dependent alternative exons. Overall design: This dataset consists of 12 RNA-seq files associated with the manuscript "Analysis of alternative exon-dependent interactome remodelling reveals multitasking functions of gene regulatory factors" characterizing the effect of Sap30bp siRNA knockdown and neural/non-neural isoform rescue on gene expression and splicing in Neuro2a cell lines. For each isoform (with or without exon 3), a total of 6 samples were analyzed including non-targeting control (NTC), Sap30bp siRNA knockdown, and siRNA knockdown with Doxycycline-induced rescue with an siRNA-resistant Sap30bp cDNA performed in two biological replicates.

可变剪接（Alternative splicing）是生物复杂性扩增的核心基础，尤其在脊椎动物神经系统中。越来越多的研究证据表明，发育阶段特异性与组织特异性的可变外显子往往调控蛋白质-蛋白质相互作用，但目前仅极少数这类事件在蛋白质层面得到了功能表征。借助亲和纯化-质谱联用（affinity purification-mass spectrometry, AP-MS）技术，我们发现：在既往被报道参与转录调控的蛋白质中，约60%经分析的神经特异性可变外显子会导致其互作蛋白组发生增加或丢失。以Chtop与Sap30bp为研究对象，二者所含的神经特异性外显子会意外重塑其与mRNA加工及转运相关互作蛋白的结合模式。Sap30bp还可通过调控长度不足100 nt的‘微小内含子’的剪接，进而控制RNA的亚细胞定位，该过程会影响转录本的核滞留。Chtop与Sap30bp的这些功能，与神经系统发育相关的转录组调控程序密切相关。因此，AP-MS技术是解析由环境依赖性可变外显子赋予蛋白质多重功能的有力工具。

整体实验设计：本数据集对应论文《可变外显子依赖型互作组重塑分析揭示基因调控因子的多任务功能》，包含12份RNA测序文件，用于表征Sap30bp小干扰RNA（small interfering RNA, siRNA）敲低以及神经/非神经亚型回复实验对Neuro2a细胞系中基因表达与剪接的影响。针对每一种亚型（含或不含外显子3），共设置6组样本进行分析，包括非靶向对照（non-targeting control, NTC）、Sap30bp siRNA敲低组，以及经强力霉素（Doxycycline）诱导表达siRNA抗性型Sap30bp cDNA以回复敲低效应的组别，所有组别均设置两次生物学重复。