Engineering circular RNA regulators to specifically promote circular RNA production.

IF 12.4 1区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL
Theranostics Pub Date : 2021-05-24 eCollection Date: 2021-01-01 DOI:10.7150/thno.56990
Yangfan Qi, Wei Han, Dan Chen, Jinyao Zhao, Lu Bai, Fang Huang, Zhenwei Dai, Gang Li, Chaoqun Chen, Wenjing Zhang, Jinrui Zhang, Bilian Jin, Yang Wang
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引用次数: 13

Abstract

Background: A large number of circular RNAs (circRNAs) have been discovered in the mammalian transcriptome with high abundance, which play vital roles in gene regulation, thereby participating in the development of multiple diseases. However, the biogenesis, regulation, and especially manipulation of circRNAs still remain largely unknown. Methods: Engineering circRNA regulators (ECRRs) were developed to promote circRNA biogenesis. Multiple circRNA mini-gene reporters were generated to evaluate the regulatory role of ECRRs. RT-PCR, qRT-PCR, northern blot, western blot, and flow cytometry assays were applied to assess the efficiency of artificial circRNA regulators on circRNA production in the presence or absence of RNase R treatment. Results: We engineered circRNA regulators by combining sequence-specific RNA binding motifs of human Pumilio 1 with functional domains that could form dimerization. We applied these engineered regulators to promote the circRNA production of the exogenous circRNA minigene reporter circGFP, thereby stimulating the functional GFP protein generation. Crucially, such regulation is in time-course dependent and dose-dependent manners with designed specificity. Moreover, the application of ECRRs could also stimulate circRNA biogenesis of another minigene reporter circScreen, suggesting that ECRRs can be commonly used to promote circRNA generation of exogenous reporters. Most importantly, ECRRs could be utilized to specifically promote the production of the endogenous circRNAs circ10720 and circBIRC6 as well. Conclusion: Our approach allows the creation of engineered regulators to target virtually any pre-mRNA in vivo, offering a novel avenue to investigate circRNA biogenesis and manipulate disease-related circRNA production.

Abstract Image

Abstract Image

Abstract Image

设计环状RNA调节因子以特异性地促进环状RNA的产生。
背景:在哺乳动物转录组中发现了大量高丰度的环状rna (circular rna, circRNAs),它们在基因调控中起着至关重要的作用,参与多种疾病的发生发展。然而,circrna的生物发生、调控,尤其是操纵在很大程度上仍然未知。方法:开发工程circRNA调节剂(ECRRs)来促进circRNA的生物发生。产生了多个circRNA迷你基因报告器来评估ecrr的调控作用。应用RT-PCR、qRT-PCR、northern blot、western blot和流式细胞术检测,评估在存在或不存在RNase R处理的情况下,人工circRNA调节剂对circRNA产生的效率。结果:我们通过将人类Pumilio 1的序列特异性RNA结合基序与可以形成二聚化的功能结构域结合来设计circRNA调节因子。我们应用这些工程化调控因子促进外源性环状rna小基因报告基因circfp的circRNA产生,从而刺激功能性GFP蛋白的产生。至关重要的是,这种调节是时间过程依赖和剂量依赖的方式,具有设计的特异性。此外,ECRRs的应用还可以刺激另一种迷你基因报告基因circScreen的circRNA生物发生,这表明ECRRs可以普遍用于促进外源性报告基因circRNA的生成。最重要的是,ECRRs还可以用来特异性地促进内源性circrna circ10720和cirbirc6的产生。结论:我们的方法允许创建工程化调节因子,在体内靶向几乎任何前mrna,为研究circRNA的生物发生和操纵疾病相关circRNA的产生提供了一种新的途径。
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来源期刊
Theranostics
Theranostics MEDICINE, RESEARCH & EXPERIMENTAL-
CiteScore
25.40
自引率
1.60%
发文量
433
审稿时长
1 months
期刊介绍: Theranostics serves as a pivotal platform for the exchange of clinical and scientific insights within the diagnostic and therapeutic molecular and nanomedicine community, along with allied professions engaged in integrating molecular imaging and therapy. As a multidisciplinary journal, Theranostics showcases innovative research articles spanning fields such as in vitro diagnostics and prognostics, in vivo molecular imaging, molecular therapeutics, image-guided therapy, biosensor technology, nanobiosensors, bioelectronics, system biology, translational medicine, point-of-care applications, and personalized medicine. Encouraging a broad spectrum of biomedical research with potential theranostic applications, the journal rigorously peer-reviews primary research, alongside publishing reviews, news, and commentary that aim to bridge the gap between the laboratory, clinic, and biotechnology industries.
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