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Dec 2020

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Quantitative Analysis of RNA Editing at Specific Sites in Plant Mitochondria or Chloroplasts Using DNA Sequencing
利用DNA测序对植物线粒体或叶绿体特定位点RNA编辑的定量分析    

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Abstract

Cytidine-to-uridine (C-to-U) RNA editing is one of the most important post-transcriptional RNA processing in plant mitochondria and chloroplasts. Several techniques have been developed to detect the RNA editing efficiency in plant mitochondria and chloroplasts, such as poisoned primer extension (PPE) assays, high-resolution melting (HRM) analysis, and DNA sequencing. Here, we describe a method for the quantitative detection of RNA editing at specific sites by sequencing cDNA from plant leaves to further evaluate the effect of different treatments or plant mutants on the C to U RNA editing in mitochondria and chloroplasts.

Keywords: C to U RNA editing (C到U的RNA编辑), DNA sequencing (DNA测序), Mitochondria (线粒体), Chloroplasts (叶绿体), Editing extent (编辑的程度)

Background

C to U RNA editing is one of the most important post-transcriptional modifications that occur in the plant mitochondrial or chloroplast genes, which usually changes the first or second positions of nucleic acid triplet codons leading to altered protein sequences and is essential for their normal functions (Takenaka et al., 2013; Yan et al., 2018). The RNA editing and processing in mitochondria or chloroplasts have been reported to function in plant male sterility, seed development, adaptations to the environment, and resistance to pathogens (Hammani et al., 2011; Dahan and Mireau, 2013; Garcia-Andrade et al., 2013; Barkan and Small, 2014; Ren et al., 2020; Yang et al., 2020). Several methods have been established for the detection of RNA editing sites or editing levels, such as poisoned primer extension (PPE) assays, high-resolution melting (HRM) analysis, and DNA sequencing (Roberson et al., 2006; Chateigner-Boutin et al., 2007; Hayes and Hanson, 2007). However, the PPE assays usually require radiolabeled oligonucleotides (Hayes and Hanson, 2007). It is hard to distinguish the editing levels at two very close editing sites using HRM assays (Chateigner-Boutin et al., 2007). Currently, DNA sequencing has been an accurate, economic, and widely used method for the RNA editing assays (Bentolila et al., 2012; Brehme et al., 2015; Yang et al., 2017; He et al., 2018). Here, we describe a method for the RNA editing detection by DNA sequencing of cDNA to further evaluate the effects of different treatments or plant mutants on the C to U RNA editing in mitochondrial and chloroplast transcripts (Yang et al., 2020).

Materials and Reagents

  1. Pipette tips and tubes (Axygen, different sizes, and types)

  2. Nicotiana benthamiana leaves

  3. TRIzolTM Reagent (Invitrogen, catalog number: 15596026)

  4. PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, catalog number: DRR047A)

  5. FastPfu DNA Polymerase (TransGen Biotech, catalog number: AP221-01)

  6. Liquid nitrogen

  7. Gel loading dye

  8. TAE buffer (Dunker et al., 2021)

  9. Agarose (Invitrogen, catalog number: 75510019)

  10. Marker III (TIANGEN Biotech, catalog number: MD103)

Equipment

  1. Thermal Cycler (Bio-Rad, model: S1000)

  2. Refrigerated Centrifuge (Thermo Fisher, model: Legend Micro 17R)

  3. Electrophoresis System (BEIJING LIUYI BIOTECHNOLOGY CO., LTD., DYY-7C)

  4. PIPETMAN Pipettes (Gilson, models: P1000, P100, P20, P2, catalog numbers: F123602, F123615, F123600, F144801)

Software

  1. BioEdit (https://bioedit.software.informer.com/)

  2. Primer Premier 5 (https://primer-premier-5.software.informer.com/)

Procedure

  1. Primer design and template generation

    1. To detect the RNA editing at a specific or several RNA editing sites in mitochondria or chloroplast transcripts, the forward and reverse primers could be designed according to sequences ~100 bp upstream and downstream of the editing sites using Primer 5 or other primer designing software (Note 1), with a recommended primer length of ~20 nt and melting temperature of ~60°C.

    2. Total RNAs from plant tissues are isolated with the TRIzolTM Reagent according to the manufacturer’s instructions and the protocols (MacRae, 2007) (Note 2).

    3. About 800 ng total RNAs were reverse-transcribed into cDNA using a PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa) according to the manufacturer’s instructions with the final volume up to 20 μl (Note 3).


  2. PCR amplification and DNA sequencing of the targeted editing sites

    1. Perform PCR amplification of the fragments that span the targeted RNA editing sites using high-fidelity DNA polymerase.

      Set up the PCR, adding the following components into a 0.2 ml PCR tube with the final volume up to 30 μl:

      ddH2O                                                    18.4 μl

      5× FastPfu Buffer                                       6 μl

      2.5 mM dNTPs                                          2.4 μl

      Forward primer (10 μM)                         0.8 μl

      Reverse primer (10 μM)                          0.8 μl

      FastPfu DNA Polymerase (2.5 U/μl)      0.6 μl

      Template                                                     1 μl

      Run PCR reactions:

      1 cycle         95°C, 2 min

      34 cycles     95°C, 20 s; 55°C, 20 s; 72°C, 20 s

      1 cycle         72°C, 2 min

    2. Perform gel electrophoresis (2% agarose) with half of the PCR products to confirm whether the amplification is specific and correct in size.

    3. Perform DNA sequencing with the other half of the PCR products using the amplification primers by a DNA sequencing service provider (Note 4).



      Figure 1. Workflow of the RNA editing analysis of specific sites in mitochondria and chloroplasts

Data analysis

As shown in Figure 1, cDNA sequences were evaluated for their respective C to T differences. The extent of RNA editing was estimated by the relative height of the respective nucleotide peaks in the sequence analysis using the BioEdit software. The editing levels are calculated as editing efficiency %= T/(C+T) ×100%.

  Here, we show an example of our previously published work (Yang et al., 2020). We analyzed the C to U RNA editing extent of several mitochondrial genes in NbMORF8-silenced N. benthamiana leaves using GFP silenced leaves as the control. We used one site as an example to indicate the quantitative measurement of the peaks using BioEdit (Figure 2).

  1. Open the sequencing files by BioEdit software.

  2. Put the black cross at the top of targeted nucleotide peaks (nucleotide T and C, respectively) and read the second number, which shows the peak height.

  3. Calculate the editing extent as efficiency %= T/(C+T) ×100%



    Figure 2. Analysis of RNA editing extent by Bioedit. The blue arrows mark the targeted nucleotide peaks, and the black hollow boxes mark the reads of the peak heights.

Notes

  1. The size of the PCR fragments is recommended in the range of 150-300 bp.

  2. The quality of the total RNA is crucial. Therefore, it is recommended to use the best RNA isolation method optimized for different plant species.

  3. Since genomic DNA sequences represent an unedited version of the targeted transcripts, it is important to erase the genomic DNA to avoid its interference with the RNA editing analyses.

  4. The PCR products are recommended to be sequenced in both forward and reverse directions since the sequencing quality from one side sometimes is not sufficient for further analysis. In our case, the DNA sequencing was conducted by a sequencing company using Applied Biosystems 3730XL.

Acknowledgments

This work was supported by China Agriculture Research System (CARS-09), National Natural Science Foundation of China (31125022 and 31930094), and the Program of Introducing Talents of Innovative Discipline to Universities (project 111) from the State of Administration for Foreign Experts Affairs, P. R. China (B18042). This protocol is adapted from Yang et al. (2020) that we previously published in Plant Physiology.

Competing interests

The authors declare no conflict of interests.

References

  1. Barkan, A. and Small, I. (2014). Pentatricopeptide repeat proteins in plants. Annu Rev Plant Biol 65: 415-442.
  2. Bentolila, S., Heller, W. P., Sun, T., Babina, A. M., Friso, G., van Wijk, K. J. and Hanson, M. R. (2012). RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing. Proc Natl Acad Sci U S A 109(22): E1453-1461.
  3. Brehme, N., Bayer-Csaszar, E., Glass, F. and Takenaka, M. (2015). The DYW Subgroup PPR Protein MEF35 Targets RNA Editing Sites in the Mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana. PLoS One 10(10): e0140680.
  4. Chateigner-Boutin, A. L. and Small, I. (2007). A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons. Nucleic Acids Res 35(17): e114.
  5. Dahan, J. and Mireau, H. (2013). The Rf and Rf-like PPR in higher plants, a fast-evolving subclass of PPR genes. RNA Biol 10(9): 1469-1476.
  6. Dunker, F., Lederer, B. and Weiberg, A. (2021). Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis. Bio Protoc 11(3): e3911.
  7. Garcia-Andrade, J., Ramirez, V., Lopez, A. and Vera, P. (2013). Mediated plastid RNA editing in plant immunity. PLoS Pathog 9(10): e1003713.
  8. Hammani, K., des Francs-Small, C. C., Takenaka, M., Tanz, S. K., Okuda, K., Shikanai, T., Brennicke, A. and Small, I. (2011). The pentatricopeptide repeat protein OTP87 is essential for RNA editing of nad7 and atp1 transcripts in Arabidopsis mitochondria. J Biol Chem 286(24): 21361-21371.
  9. Hayes, M. L. and Hanson, M. R. (2007). Assay of editing of exogenous RNAs in chloroplast extracts of Arabidopsis, maize, pea, and tobacco. Methods Enzymol 424: 459-482.
  10. He, P., Xiao, G., Liu, H., Zhang, L., Zhao, L., Tang, M., Huang, S., An, Y. and Yu, J. (2018). Two pivotal RNA editing sites in the mitochondrial atp1mRNA are required for ATP synthase to produce sufficient ATP for cotton fiber cell elongation. New Phytol 218(1): 167-182.
  11. MacRae, E. (2007). Extraction of plant RNA. Methods Mol Biol 353: 15-24.
  12. Ren, R. C., Wang, L. L., Zhang, L., Zhao, Y. J., Wu, J. W., Wei, Y. M., Zhang, X. S. and Zhao, X. Y. (2020). DEK43 is a P-type pentatricopeptide repeat (PPR) protein responsible for the Cis-splicing of nad4 in maize mitochondria. J Integr Plant Biol 62(3): 299-313.
  13. Roberson, L. M. and Rosenthal, J. J. (2006). An accurate fluorescent assay for quantifying the extent of RNA editing. RNA 12(10): 1907-1912.
  14. Takenaka, M., Zehrmann, A., Verbitskiy, D., Hartel, B. and Brennicke, A. (2013). RNA editing in plants and its evolution. Annu Rev Genet 47: 335-352.
  15. Yan, J., Zhang, Q. and Yin, P. (2018). RNA editing machinery in plant organelles. Sci China Life Sci 61(2): 162-169.
  16. Yang, Y., Fan, G., Zhao, Y., Wen, Q., Wu, P., Meng, Y. and Shan, W. (2020). Cytidine-to-Uridine RNA Editing Factor NbMORF8 Negatively Regulates Plant Immunity to Phytophthora Pathogens. Plant Physiol 184(4): 2182-2198.
  17. Yang, Y., Zhu, G., Li, R., Yan, S., Fu, D., Zhu, B., Tian, H., Luo, Y. and Zhu, H. (2017). The RNA Editing Factor SlORRM4 Is Required for Normal Fruit Ripening in Tomato. Plant Physiol 175(4): 1690-1702.

简介

[摘要]胞苷-尿苷(C-to-U)RNA编辑是植物线粒体和叶绿体中最重要的转录后RNA加工之一。已经开发了多种技术来检测植物线粒体和叶绿体中的 RNA 编辑效率,例如中毒引物延伸 (PPE) 分析、高分辨率熔解 (HRM) 分析和 DNA 测序。在这里,我们描述了一种通过对植物叶子的 cDNA 进行测序来定量检测特定位点 RNA 编辑的方法,以进一步评估不同处理或植物突变体对线粒体和叶绿体中 C 到 U RNA 编辑的影响。


[背景] C to U RNA 编辑是最重要的编辑之一 转录后修饰小号发生在植物线粒体或叶绿体基因,这通常改变核酸三联密码子,导致改变的蛋白质序列的第一或第二位置,是必需对它们的正常功能(竹中等人,2013;颜等人, 201 8 ) 。据报道,线粒体或叶绿体中的 RNA 编辑和加工在植物雄性不育、种子发育、对环境的适应和对病原体的抗性中发挥作用(Hammani等,2011;Dahan和 Mireau ,2013;Garcia-Andrade等) . , 2013; Barkan and Small , 2014 ; Ren et al. , 20 20 ; Yang et al. , 2020) 。已经建立了几种检测 RNA 编辑位点或编辑水平的方法,例如中毒引物延伸 (PPE) 分析、高分辨率熔解 (HRM) 分析和 DNA 测序(Roberson等人,2006 年;Chateigner-Boutin等人) al. , 2007; Hayes和 Hanson , 2007) 。然而,PPE 分析通常需要放射性标记的寡核苷酸(Hayes和 Hanson ,2007)。使用 HRM 分析很难区分两个非常接近的编辑位点的编辑水平(Chateigner-Boutin等,2007)。目前,DNA 测序已成为 RNA 编辑分析中一种准确、经济且广泛使用的方法(Bentolila等,2012;Brehme等,2015;Yang等,2017 ;He等,2018)。在这里,我们描述了一种通过 cDNA 的 DNA 测序进行 RNA 编辑检测的方法,以进一步评估不同处理或植物突变体对线粒体和叶绿体转录物中C 到 U RNA 编辑的影响(Yang等,2020)。

关键字:C到U的RNA编辑, DNA测序, 线粒体, 叶绿体, 编辑的程度

材料和试剂
 
1.移液管头和管(爱思进,不同尺寸,和类型)      
2.本氏烟草叶      
3. TRIzol TM Reagent(Invitrogen,目录号:15596026)      
4. PrimeScript RT Reagent Kit with gDNA Eraser(Perfect Real Time)(Ta K aRa,目录号:DRR047A)      
5. FastPfu DNA聚合酶(TransGen Biotech,目录号:AP221-01)      
6.液氮      
7. ģ EL样染料      
8. TAE 缓冲液(Dunker等人,2021 年)      
9.琼脂糖(Invitrogen,目录号:75510019)      
10. Marker III(TIANGEN Biotech,目录号:MD103)   
 
设备
 
热循环仪(B io -R ad ,型号:S1000 )
冷冻离心机(Thermo Fisher,型号:Legend Micro 17R )
电泳系统(北京六一生物科技有限公司,DYY-7C)
的Pipetman移液管(吉尔森,型号小号:P1000,P100,P20,P2 ,目录号小号:F123602,F123615,F123600,F144801)
 
软件
 
BioEdit ( https://bioedit.software.informer.com/ )
Primer Premier 5 ( https://primer-premier-5.software.informer.com/)
 
程序
 
引物设计和模板生成
为了检测线粒体或叶绿体转录物中特定或多个 RNA 编辑位点的RNA 编辑,可以使用 Primer 5 或其他引物设计软件,根据编辑位点上游和下游约 100 bp 的序列设计正向和反向引物(注1),具有一个推荐〜20个核苷酸和〜60的熔融温度的引物长度℃下。
根据制造商的说明和方案(MacRae,2007)(注 2),使用TRIzol TM试剂从植物组织中分离总 RNA 。
根据制造商的说明,使用 PrimeScript RT Reagent Kit 和 gDNA Eraser (Perfect Real Time) ( Ta K aRa ) 将大约 800 ng 总 RNA 逆转录为 cDNA ,最终体积高达 20 μl (注 3)。
 
目标编辑位点的 PCR 扩增和 DNA 测序
使用高保真 DNA 聚合酶对跨越目标 RNA 编辑位点的片段进行 PCR 扩增。
设置 PCR,将以下组分添加到 0.2 ml 的PCR 管中,最终体积可达 30 μl :
ddH 2 O 18.4 μ l
5×FastPfu 缓冲液 6 μl
2.5 mM dNTP 2.4 μ l
正向引物 (10 μM) 0.8 μl
反向引物 (10 μM) 0.8 μl
FastPfu DNA 聚合酶 (2.5 U/μ l ) 0.6 μ l
模板 1 μ l
运行 PCR 反应:
1 个循环 95°C,2 分钟
34 个循环 95°C,20 秒;55°C,20 秒;72°C,20 秒
1 个循环 72 °C,2 分钟
用一半的 PCR 产物进行凝胶电泳(2% 琼脂糖),以确认扩增是否特异且大小正确。
使用 DNA 测序服务提供商提供的扩增引物对另一半 PCR 产物进行 DNA 测序(注 4)。
 
 
图 1.线粒体和叶绿体特定位点 RNA 编辑分析的工作流程
 
数据分析
 
如图 1 所示,评估了 cDNA 序列的 C 到 T 差异。RNA 编辑的程度通过使用 BioEdit 软件进行的序列分析中各个核苷酸峰的相对高度来估计。编辑水平计算为编辑效率 %= T/(C+T) × 100%。
  在这里,我们展示了我们之前发表的作品的一个例子(Yang等,2020)。我们分析了C到的几个线粒体基因üRNA编辑程度NbMORF8沉默的烟草本塞姆氏利用GFP沉默的叶与叶的控制。我们以一个站点为例来说明使用 BioEdit 对峰进行的定量测量(图 2)。
通过 BioEdit 软件打开测序文件。
将黑色十字放在目标核苷酸峰(分别为核苷酸 T 和 C)的顶部并读取第二个数字,该数字显示了峰高。
计算编辑程度为效率%= T/(C+T) × 100%
 
 
图 2. Bioedit 对 RNA 编辑范围的分析。蓝色箭头标记目标核苷酸峰,黑色空心框标记峰高读数。
 
笔记
 
建议 PCR 片段的大小在 150 - 300 bp范围内。
总 RNA 的质量至关重要。因此,建议使用针对不同植物物种优化的最佳 RNA 分离方法。
由于基因组DNA的序列代表一个目标转录物的未经编辑的版本,它擦除的基因组DNA,以避免其干扰是很重要的与所述RNA编辑的分析。
建议对 PCR 产物进行正向和反向测序,因为一侧的测序质量有时不足以进行进一步分析。在我们的案例中,DNA 测序是由一家测序公司使用 Applied Biosystems 3730XL 进行的。
 
致谢
 
该工作得到了中国农业研究系统(CARS-09)、国家自然科学基金(31125022和31930094)、国家外国专家局创新学科人才引进计划(111项目)的支持中国事务 (B18042)。该协议改编自杨等人。(2020)我们之前发表在植物生理学上。
 
利益争夺
 
作者宣称没有利益冲突小号。
 
参考
 
Barkan, A. 和 Small, I. (2014)。植物中的五肽重复蛋白。Annu Rev Plant Biol 65:415-442。
Bentolila, S., Heller, WP, Sun, T., Babina, AM, Friso, G., van Wijk, KJ 和 Hanson, MR (2012)。RIP1 是拟南芥蛋白家族的成员,与蛋白 RARE1 相互作用并广泛影响 RNA 编辑。Proc Natl Acad Sci USA 109(22): E1453-1461。
Brehme, N.、Bayer-Csaszar, E.、Glass, F. 和 Takenaka, M.(2015 年)。DYW 亚群 PPR 蛋白 MEF35 靶向拟南芥中线粒体 rpl16、nad4 和 cob mRNA 中的 RNA 编辑位点。PLoS 一10(10):e0140680。
Chateigner-Boutin, AL 和 Small, I. (2007)。基于扩增子高分辨率熔解的 RNA 编辑检测和定量快速高通量方法。核酸研究35(17):e114。
Dahan, J. 和 Mireau, H.(2013 年)。高等植物中的 Rf 和 Rf 样 PPR,一个快速进化的 PPR 基因亚类。RNA 生物学10(9):1469-1476。
Dunker, F.、Lederer, B. 和 Weiberg, A.(2021 年)。用于病原体跨界小 RNA 分析的植物 ARGONAUTE 蛋白质免疫纯化。生物协议11(3):e3911。              
Garcia-Andrade, J.、Ramirez, V.、Lopez, A. 和 Vera, P. (2013)。植物免疫中介导的质体 RNA 编辑。PLoS 病原体9(10):e1003713。
Hammani, K., des Francs-Small, CC, Takenaka, M., Tanz, SK, Okuda, K., Shikanai, T., Brennicke, A. 和 Small, I. (2011)。五肽重复蛋白 OTP87 对于拟南芥线粒体中 nad7 和 atp1 转录本的 RNA 编辑至关重要。J Biol Chem 286(24): 21361-21371。
Hayes, ML 和 Hanson, MR (2007)。拟南芥、玉米、豌豆和烟草叶绿体提取物中外源 RNA 的编辑分析。方法 Enzymol 424:459-482。              
He, P., Xiao, G., Liu, H., Zhang, L., Zhao, L., Tang, M., Huang, S., An, Y. 和 Yu, J. (2018)。ATP 合酶需要线粒体 atp1mRNA 中的两个关键 RNA 编辑位点,以产生足够的 ATP 用于棉花纤维细胞的伸长。新植醇218(1): 167-182。
麦克雷,E.(2007 年)。植物RNA的提取。方法 Mol Biol 353:15-24。
Ren, RC, Wang, LL, Zhang, L., Zhao, YJ, Wu, JW, Wei, YM, Zhang, XS 和 Zhao, XY (2020)。DEK43 是一种 P 型五肽重复序列 (PPR) 蛋白,负责玉米线粒体中 nad4 的顺式剪接。J Integr Plant Biol 62(3): 299-313。
Roberson, LM 和 Rosenthal, JJ (2006)。用于量化 RNA 编辑程度的准确荧光检测。RNA 12(10):1907-1912。              
Takenaka, M.、Zehrmann, A.、Verbitskiy, D.、Hartel, B. 和 Brennicke, A. (2013)。植物中的 RNA 编辑及其进化。Annu Rev Genet 47:335-352。
Yan, J.、Zhang, Q. 和 Yin, P. (2018)。植物细胞器中的 RNA 编辑机制。科学 中国人寿科学61(2): 162-169。
Yang, Y.、Fan, G.、Zhao, Y.、Wen, Q.、Wu, P.、Meng, Y. 和 Shan, W. (2020)。胞苷-尿苷 RNA 编辑因子 NbMORF8 消极调节植物对疫霉病病原体的免疫力。植物生理学184(4): 2182-2198。
Yang, Y., Zhu, G., Li, R., Yan, S., Fu, D., Zhu, B., Tian, H., Luo, Y. 和 Zhu, H. (2017)。RNA 编辑因子 SlORRM4 是番茄果实正常成熟所必需的。植物生理学175(4): 1690-1702。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yang, Y. and Shan, W. (2021). Quantitative Analysis of RNA Editing at Specific Sites in Plant Mitochondria or Chloroplasts Using DNA Sequencing. Bio-protocol 11(18): e4154. DOI: 10.21769/BioProtoc.4154.
  2. Yang, Y., Fan, G., Zhao, Y., Wen, Q., Wu, P., Meng, Y. and Shan, W. (2020). Cytidine-to-Uridine RNA Editing Factor NbMORF8 Negatively Regulates Plant Immunity to Phytophthora Pathogens. Plant Physiol 184(4): 2182-2198.
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