RNAi Screening to Identify Factors That Control Circular RNA Localization.
RNAi Screening to Identify Factors That Control Circular RNA Localization.
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DOI：
10.1007/978-1-0716-0935-4_20
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发表时间：
2021
影响因子：
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通讯作者：
Deirdre C. Tatomer;Dongming Liang;J. Wilusz
中科院分区：
文献类型：
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作者：
Deirdre C. Tatomer;Dongming Liang;J. Wilusz
数以千计的真核蛋白质编码基因被非规范地拼接起来，产生具有共价连接末端的环状RNA。这些转录本对核酸外切酶的降解具有抵抗力，这使一些转录本能够积累到比相关的线性mRNA更高的水平。一般来说，外显子环状RNA聚集在细胞质中，但大多数转录本的功能尚不清楚。已有的研究表明，一些蛋白质可以调节microRNAs或RNA结合蛋白的活性，被翻译成蛋白质产物，或者调节先天免疫反应。最近的工作表明，环状RNA以一种长度依赖的方式从细胞核输出，这些转录本的亚细胞定位可以由果蝇中的DExH/D-box解旋酶Hel25E控制。在这里，我们描述了如何使用RNAi筛选结合亚细胞分离和定量逆转录聚合酶链式反应(RT-qPCR)来识别果蝇细胞中环状RNA定位的调控因子。激活RNA干扰(RNAi)途径的长双链RNA(DsRNAs)被用来耗尽感兴趣的因子，然后用生化分级来分离核质RNA。然后，用RT-qPCR引物在特定环状RNA的后剪接连接处扩增，以定量这些转录本在核间和细胞质间的相对数量。总而言之，该方法可广泛用于表征环状RNA核输出和本地化机制，包括识别新的调控因子及其环状RNA靶标的广度。
Thousands of eukaryotic protein-coding genes are noncanonically spliced to generate circular RNAs that have covalently linked ends. These transcripts are resistant to degradation by exonucleases, which enables some to accumulate to higher levels than the associated linear mRNA. In general, exonic circular RNAs accumulate in the cytoplasm, but functions for most of these transcripts remain unknown. It has been proposed that some may modulate the activity of microRNAs or RNA-binding proteins, be translated to yield protein products, or regulate innate immune responses. Recent work has revealed that circular RNAs are exported from the nucleus in a length-dependent manner and that the subcellular localization of these transcripts can be controlled by the DExH/D-box helicase Hel25E inDrosophila. Here, we describe how RNAi screening combined with subcellular fractionation and quantitative reverse transcription PCR (RT-qPCR) can be used to identify regulators of circular RNA localization inDrosophilacells. Long double-stranded RNAs (dsRNAs) that activate the RNA interference (RNAi) pathway are used to deplete factors of interest followed by biochemical fractionation to separate nuclear and cytoplasmic RNAs. RT-qPCR primers that amplify across the backsplicing junction of specific circular RNAs are then used to quantify the relative amounts of these transcripts in the nuclear and cytoplasmic compartments. In total, this approach can be broadly used to characterize circular RNA nuclear export and localization mechanisms, including to identify novel regulatory factors and their breadth of circular RNA targets.