Department of Biochemistry & Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
Methods Mol Biol. 2021;2209:321-332. doi: 10.1007/978-1-0716-0935-4_20.
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 in Drosophila. 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 in Drosophila cells. 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.
数以千计的真核生物蛋白编码基因发生非规范剪接，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抗性，这使得一些转录本的积累水平高于相关的线性 mRNA。通常，外显子环状 RNA 在细胞质中积累，但这些转录本的大多数功能仍然未知。有人提出，一些环状 RNA 可能调节 microRNA 或 RNA 结合蛋白的活性，被翻译产生蛋白质产物，或调节先天免疫反应。最近的研究工作表明，环状 RNA 以依赖于长度的方式从核内输出，并且这些转录本的亚细胞定位可以被果蝇中的 DExH/D 盒解旋酶 Hel25E 控制。在这里，我们描述了如何使用 RNAi 筛选结合亚细胞分级分离和定量反转录 PCR（RT-qPCR）来鉴定果蝇细胞中环状 RNA 定位的调节剂。激活 RNA 干扰 (RNAi) 途径的长双链 RNA (dsRNA) 用于耗尽感兴趣的因子，然后进行生化分级分离以分离核和细胞质 RNA。然后使用跨越特定环状 RNA 回文拼接连接的 RT-qPCR 引物来定量这些转录本在核和细胞质区室中的相对含量。总的来说，这种方法可以广泛用于表征环状 RNA 的核输出和定位机制，包括鉴定新的调节因子及其环状 RNA 靶标的广泛范围。
