Summary

使用含有微型基因的Alu元素来分析圆形RNA

Published: March 10, 2020
doi:

Summary

我们克隆和分析生成圆形RNA的记者基因。这些报告基因大于构造,用于分析线性拼接并包含Alu元素。为了研究圆形RNA,构造被转染到细胞中,在去除线性RNA后使用RT-PCR分析产生的RNA。

Abstract

除了线性 mRNA 之外,许多真核基因产生圆形 RNA。大多数圆形RNA是通过将5’拼接位点与上游3’拼接位点在mRNA前结合而生成的,这个过程称为回拼接。这种循环可能得益于mRNA前的二级结构,这些结构使拼接点非常接近。在人类基因中,Alu元素被认为能促进这些二次RNA结构,因为Alu元素非常丰富,并且在mRNA前以相反方向存在时,表现出彼此的基础互补性。在这里,我们描述了大型Alu元素的生成和分析,该元素包含构成圆形RNA的记者基因。通过优化克隆协议,可以生成插入长度高达20kb的分子基因。他们在联合转染实验中的分析可以识别监管因素。因此,该方法可以识别与循环RNA形成有关的RNA序列和细胞成分。

Introduction

循环 RNA
圆形RNA(环RNA)是同价关闭的单滞留RNA,在大多数生物体中表达。它们通过将下游5’拼接站点连接到上游3’拼接站点生成,这个过程称为反拼接(图1A)1在mRNA前序列,显示基础互补短为30-40nt,使回拼接位点在适当的对齐循环RNA形成2。在人类中,Alu元素1,约占基因组3的11%,由于自身互补4、5,在mRNA前形成广泛的双链RNA结构,从而促进RNA1的形成。

目前,已经描述了环环境的三大功能。一些环RNA结合微RNA(miRNA),并通过封存行为像miRNA海绵6。循环RNA已涉及转录和后转录调节,通过竞争与线性拼接7或修改转录因子活动8。最后,循环RNA包含短的开放阅读框架和原理证明研究表明,他们可以翻译9,10。然而,大多数环苯A的功能仍然是神秘的。大多数循环RNA已经使用下一代测序方法11检测到。使用有针对性的RT-PCR方法对单个基因进行详细分析后发现,仍有大量圆形RNA有待发现。

使用记者基因分析mRNA前处理
从DNA报告器中提取的mRNA分析构造转染成细胞是研究替代预mRNA拼接的成熟方法,可应用于圆形RNA。一般来说,替代外子、其周围突长和构成外子被放大并克隆成真核表达向量。通常,缩内缩器会缩短。这些结构被转染成真核细胞,通常由RT-PCR13,14进行分析。该方法已被广泛用于绘制监管拼接站点和转效因子在联合转染实验13,15,16,17,18。此外,蛋白质表达微型基因的产生允许筛选改变替代拼接19,20的物质。

该方法已应用于圆形RNA。目前,至少有12个微型基因骨干在文献中进行了描述,并被汇总在表1中。除了基于tRNA的表达系统21,22,他们都依赖于聚合酶II启动子。在这里,我们描述了一种生成人类报告器微基因的方法,以确定循环RNA生成所涉及的cis和跨作用因子。图1显示了使用已发布报告基因23序列的方法的概述。

Protocol

1. 结构设计 使用UCSC基因组浏览器24来识别循环RNA形成所需的重复元素,并将其纳入结构。重要的是,扩增的引素需要超出重复元素。 将圆形RNA序列(补充图1是测试序列)粘贴到https://genome.ucsc.edu/cgi-bin/hgBlat?command=start并选择正确的有机体。提交序列并转到浏览器视图,缩小 1.5 倍或根据需?…

Representative Results

报告基因允许确定影响循环RNA形成的调节因子。然而,这些报告基因很大,并且含有经常使DNA构造不稳定等重复性元素。由于其大尺寸,通常需要删除部分的内科,这是通过放大基因组片包含外子和较小的侧翼在电子部分。这些DNA片段是酶组装的,允许结构不受限制的酶。 从微管相关蛋白tau (MAPT) 生成的圆形RNA示例显示了微基因方法分析圆形RNA的应用。本例中使用的tau 9_12…

Discussion

一般来说,圆形RNA是低丰1,这使得研究其功能和形成复杂化。与线性RNA13类似,使用报告器微基因可以识别调节圆形RNA形成的cis和跨作用因子。因此,这种方法产生假设,可以使用内源基因进一步测试。

最关键的步骤是记者基因的设计。DNA片段的酶组装(”吉布森克隆“27)有助于这种设计,因为它允许构建独立于限制位点?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国防部国防部授予AZ180075的支持。斯特凡·斯塔姆感谢杰奎琳·努南捐赠。安娜·帕卢钦得到了德国学术交流项目DAAD的支持,贾斯汀·韦尔登是肯塔基大学马克斯·斯特克勒奖的获得者。

Materials

(PEI) Hydrochloride Polysciences 24765-1
Builder tool NEB https://nebuilder.neb.com/#!/
Dark Reader Transilluminator. Clare Chemical Research
Enzymatic DNA assembly kit NEB E2621S
Gel and PCR cleanup kit Promega A9282
Glyco Blue Thermo Fisher AM9516
pcDNA3.1 cloning site Polycloning site https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf
Polymerase 1 NEB M0491L Q5 DNA polymerase
Polymerase 2 Biorad 1725310 Long range polymerase (NEB), iproof (BioRad)
Polymerase 2 Qiagen 206402 Qiagen long range polymerase kit
Reverse Transcriptase Thermo Fisher 18080044
RNA isolation kit Life Technologies 12183025 Ambion by Life Technologies
RNAse R Lucigen RNR07250 Epicenter/Lucigen
Stable competent cells NEB C3040H NEB stable cells
Standard cloning bacteria NEB C2988J NEB5-alpha competent
Web tool to design primers NEB https://nebuilder.neb.com/#!/
Web-based temperature calculations NEB https://tmcalculator.neb.com/#!/main

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Diesen Artikel zitieren
Welden, J. R., Pawluchin, A., van Doorn, J., Stamm, S. Use of Alu Element Containing Minigenes to Analyze Circular RNAs. J. Vis. Exp. (157), e59760, doi:10.3791/59760 (2020).

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