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?…
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 |
Referenzen
- Jeck, W. R., et al. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA. 19 (2), 141-157 (2013).
- Zhang, X. O., et al. Complementary sequence-mediated exon circularization. Cell. 159 (1), 134-147 (2014).
- Deininger, P. Alu elements: know the SINEs. Genome Biology. 12 (12), 236 (2011).
- Bazak, L., Levanon, E. Y., Eisenberg, E. Genome-wide analysis of Alu editability. Nucleic Acids Research. 42 (11), 6876-6884 (2014).
- Levanon, E. Y., et al. Systematic identification of abundant A-to-I editing sites in the human transcriptome. Nature Biotechnology. 22 (8), 1001-1005 (2004).
- Hansen, T. B., et al. Natural RNA circles function as efficient microRNA sponges. Nature. 495 (7441), 384-388 (2013).
- Kelly, S., Greenman, C., Cook, P. R., Papantonis, A. Exon Skipping Is Correlated with Exon Circularization. Journal of Molecular Biology. 427 (15), 2414-2417 (2015).
- Li, Z., et al. Exon-intron circular RNAs regulate transcription in the nucleus. Nature Structural & Molecular Biology. 22 (3), 256-264 (2015).
- Yang, Y., et al. Extensive translation of circular RNAs driven by N(6)-methyladenosine. Cell Research. 27 (6), 626-641 (2017).
- Abe, N., et al. Rolling Circle Translation of Circular RNA in Living Human Cells. Scientific Reports. 5, 16435 (2015).
- Salzman, J., Chen, R. E., Olsen, M. N., Wang, P. L., Brown, P. O. Cell-type specific features of circular RNA expression. PLOS Genetics. 9 (9), 1003777 (2013).
- Ottesen, E. W., Luo, D., Seo, J., Singh, N. N., Singh, R. N. Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs. Nucleic Acids Research. 47 (6), 2884-2905 (2019).
- Stoss, O., Stoilov, P., Hartmann, A. M., Nayler, O., Stamm, S. The in vivo minigene approach to analyze tissue-specific splicing. Brain Research Protocols. 4, 383-394 (1999).
- Mardon, H. J., Sebastio, G., Baralle, F. E. A role for exon sequences in alternative splicing of the human fibronectin gene. Nucleic Acids Research. 15, 7725-7733 (1987).
- Gaildrat, P., et al. Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants. Methods in Molecular Biology. 653, 249-257 (2010).
- Cooper, T. A. Use of minigene systems to dissect alternative splicing elements. Methods. 37 (4), 331-340 (2005).
- Baralle, D., Baralle, M. Splicing in action: assessing disease causing sequence changes. Journal of Medical Genetics. 42 (10), 737-748 (2005).
- Percifield, R., Murphy, D., Stoilov, P. Medium throughput analysis of alternative splicing by fluorescently labeled RT-PCR. Methods in Molecular Biology. 1126, 299-313 (2014).
- Stoilov, P., Lin, C. H., Damoiseaux, R., Nikolic, J., Black, D. L. A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators. Proceedings of the National Academy of Sciences of the United States of America. 105 (32), 11218-11223 (2008).
- Shen, M., et al. Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure. Nucleic Acids Research. 41 (6), 3819-3832 (2013).
- Noto, J. J., Schmidt, C. A., Matera, A. G. Engineering and expressing circular RNAs via tRNA splicing. RNA Biology. , 1-7 (2017).
- Schmidt, C. A., Noto, J. J., Filonov, G. S., Matera, A. G. A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing. Methods in Enzymology. 572, 215-236 (2016).
- Welden, J. R., van Doorn, J., Nelson, P. T., Stamm, S. The human MAPT locus generates circular RNAs. Biochimica et Biophysica Acta – Molecular Basis of Disease. , 2753-2760 (2018).
- Casper, J., et al. The UCSC Genome Browser database: 2018 update. Nucleic Acids Research. 46, 762-769 (2018).
- Grozdanov, P. N., MacDonald, C. C. Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1alpha promoter using Gibson assembly. Journal of Visualized Experiments. (96), (2015).
- Wang, Y., et al. A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Research. 32 (3), 1197-1207 (2004).
- Gibson, D. G., et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. 6 (5), 343-345 (2009).
- Conn, S. J., et al. The RNA binding protein quaking regulates formation of circRNAs. Cell. 160 (6), 1125-1134 (2015).
- Jeck, W. R., Sharpless, N. E. Detecting and characterizing circular RNAs. Nature Biotechnology. 32 (5), 453-461 (2014).
- Pamudurti, N. R., et al. Translation of CircRNAs. Molecular Cell. 66 (1), 9-21 (2017).
- Kramer, M. C., et al. Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins. Genes & Development. 29 (20), 2168-2182 (2015).
- Starke, S., et al. Exon circularization requires canonical splice signals. Cell Reports. 10 (1), 103-111 (2015).
- Suzuki, H., Tsukahara, T. A view of pre-mRNA splicing from RNase R resistant RNAs. International Journal of Molecular Sciences. 15 (6), 9331-9342 (2014).
- Zhang, X. O., et al. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs. Genome Research. 26 (9), 1277-1287 (2016).
- Liang, D., Wilusz, J. E. Short intronic repeat sequences facilitate circular RNA production. Genes & Development. 28 (20), 2233-2247 (2014).
- Wang, Y., Mandelkow, E. Tau in physiology and pathology. Nature Reviews Neuroscience. 17 (1), 5-21 (2016).
- Fejes-Toth, K., et al. Post-transcriptional processing generates a diversity of 5′-modified long and short RNAs. Nature. 457 (7232), 1028-1032 (2009).
- Gao, Q. S., et al. Complex regulation of tau exon 10, whose missplicing causes frontotemporal dementia. Journal of Neurochemistry. 74 (2), 490-500 (2000).
- Hartmann, A. M., et al. Regulation of alternative splicing of human tau exon 10 by phosphorylation of splicing factors. Molecular and Cellular Neuroscience. 18 (1), 80-90 (2001).
- Wang, Y., Wang, Z. Efficient backsplicing produces translatable circular mRNAs. RNA. 21 (2), 172-179 (2015).
- Yang, Y., Wang, Z. Constructing GFP-Based Reporter to Study Back Splicing and Translation of Circular RNA. Methods in Molecular Biology. 1724, 107-118 (2018).
- Zheng, Q., et al. Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nature Communications. 7, 11215 (2016).
- Jia, W., Xu, B., Wu, J. Circular RNA expression profiles of mouse ovaries during postnatal development and the function of circular RNA epidermal growth factor receptor in granulosa cells. Metabolism. 85, 192-204 (2018).
- Liang, D., et al. The Output of Protein-Coding Genes Shifts to Circular RNAs When the Pre-mRNA Processing Machinery Is Limiting. Molecular Cell. 68 (5), 940-954 (2017).
- Legnini, I., et al. Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis. Molecular Cell. 66 (1), 22-37 (2017).
- Li, X., et al. Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection. Molecular Cell. 67 (2), 214-227 (2017).
- Zhang, Y., et al. The Biogenesis of Nascent Circular RNAs. Cell Reports. 15 (3), 611-624 (2016).
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).