A Cell-Based High-Throughput Method for Identifying Modulators of Alternative Splicing.
A Cell-Based High-Throughput Method for Identifying Modulators of Alternative Splicing.
复制标题
一种基于细胞的高通量方法，用于识别选择性剪接的调节剂。
DOI：
10.1007/978-1-4939-7204-3_16
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Zheng,Sika
中科院分区：
文献类型：
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作者：
Zheng,Sika
选择性剪接是基因表达的一个重要调控过程，它受反式作用因子的控制，通过识别RNA转录产物中的顺式元件来影响剪接体的组装和剪接位点的选择。细胞外刺激和信号级联可以会聚在RNA结合剪接调节子上以影响选择性剪接。剪接调控的缺陷与各种人类疾病有关，并且引起疾病的剪接事件的修饰呈现出巨大的治疗前景。由于低通量、低灵敏度和低特异性，确定剪接调节子和/或上游调节子是困难的。IRAS（identifyingregulators ofalternativesplicing）是一种新的基于细胞的高通量筛选策略，专门针对这些挑战而设计，并实现了高通量、高灵敏度和高特异性。在这里，我们详细描述了IRAS方法与一对双荧光小基因报告，产生GFP和RFP荧光信号，以测定两个剪接异构体排他。这两个互补的微型基因报告改变GFP/RFP输出比例在相反的方向，在响应只有一个真正的剪接变化。来自信号筛选的假阳性不会刺激GFP/RFP比率的相反变化。报告基因对与机器人液体处理器和阵列文库相结合，允许IRAS以高通量方式筛选具有高灵敏度和特异性的阳性和阴性剪接调节子。
Alternative splicing, a key regulatory process of gene expression, is controlled bytrans-acting factors that recognizecis-elements in premature RNA transcripts to affect spliceosome assembly and splice site choices. Extracellular stimuli and signaling cascades can converge on RNA binding splicing regulators to affect alternative splicing. Defects in splicing regulation have been associated with various human diseases, and modification of disease-causing splicing events presents great therapeutic promise. Determining splicing regulators and/or upstream modulators has been difficult with low throughput, low sensitivity, and low specificity. IRAS (identifyingregulators ofalternativesplicing) is a novel cell-based high-throughput screening strategy designed specifically to address these challenges and has achieved high throughput, high sensitivity, and high specificity. Here, we describe the IRAS method in detail with a pair of dual-fluorescence minigene reporters that produces GFP and RFP fluorescent signals to assay the two spliced isoforms exclusively. These two complementary mini-gene reporters alter GFP/RFP output ratios in the opposite direction in response to only a true splicing change. False positives from a signal screen do not stimulate opposite changes in GFP/RFP ratios. The reporter pair in conjunction with robotic liquid handlers and arrayed libraries allows IRAS to screen for both positive and negative splicing regulators with high sensitivity and specificity in a high-throughput manner.