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. 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)是驱动生物复杂性扩张的核心机制，尤其在脊椎动物神经系统中。越来越多的证据表明，发育阶段依赖和组织特异性的可变外显子通常调控蛋白质-蛋白质相互作用，但目前仅少数这类事件在蛋白质层面得到了表征。
我们通过亲和纯化-质谱联用(affinity purification-mass spectrometry, AP-MS)技术发现，在先前被证实参与转录调控的蛋白质中，约60%经分析的神经差异外显子会导致互作伴侣的获得或丢失。
本研究聚焦Chtop与Sap30bp，这两种蛋白的神经特异性外显子会意外重塑其与mRNA加工及转运相关伴侣的相互作用。Sap30bp还可通过调控长度小于100 nt的“微型内含子(mini-introns)”的剪接，进而控制RNA定位，此类内含子可促进转录本的核滞留。
Chtop与Sap30bp的上述功能与神经系统发育相关的转录组调控程序紧密关联。因此，AP-MS是解析由环境依赖性可变外显子赋予蛋白质多方面功能的有力研究手段。
本数据集包含与论文《分析可变外显子依赖的互作组重塑揭示基因调控因子的多任务功能》("Analysis of alternative exon-dependent interactome remodelling reveals multitasking functions of gene regulatory factors")相关的12份RNA测序文件，该论文表征了Sap30bp小干扰RNA(small interfering RNA, siRNA)敲低以及神经/非神经亚型拯救对Neuro2a细胞系中基因表达与剪接的影响。
针对每一种亚型（带有或不带有外显子3），共分析了6份样本，包括非靶向对照(non-targeting control, NTC)、Sap30bp siRNA敲低组，以及用强力霉素(Doxycycline)诱导表达siRNA抗性Sap30bp互补DNA(complementary DNA, cDNA)进行拯救的siRNA敲低组，上述实验均设置2次生物学重复。