使用含有微型基因的Alu元素来分析圆形RNA
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，从而促进环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?…
Disclosures
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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Cite This Article
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).