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Export of transcribed mRNAs from nucleoplasm to cytosol is an essential process for the translation of genes into proteins. This process is tightly regulated by nuclear pores, composed of about 30 nucleoporin proteins (Nups). Whether or not the mRNAs are able to be appropriately exported to cytoplasm is of an importance for understanding the role of Nups. Here, we describe a practical protocol to detect the intracellular localization of mRNAs in mesophyll cells of Nicotiana benthamiana (N. benthamiana). This protocol is based on poly (A) in situ hybridization method using an oligo d(T) probe conjugated with Alexa Fluor-488.

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Detection of Poly (A) RNA in Mesophyll Cells of Nicotiana benthamiana Using in situ Hybridization
原位杂交技术检测本氏烟草叶肉细胞中的Poly(A)RNA

植物科学 > 植物细胞生物学 > 细胞染色
作者: Yuri Mizuno
Yuri MizunoAffiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
Bio-protocol author page: a2511
 and Daigo Takemoto
Daigo TakemotoAffiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
For correspondence: dtakemo@agr.nagoya-u.ac.jp
Bio-protocol author page: a2512
Vol 5, Iss 17, 9/5/2015, 1537 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1576

[Abstract] Export of transcribed mRNAs from nucleoplasm to cytosol is an essential process for the translation of genes into proteins. This process is tightly regulated by nuclear pores, composed of about 30 nucleoporin proteins (Nups). Whether or not the mRNAs are able to be appropriately exported to cytoplasm is of an importance for understanding the role of Nups. Here, we describe a practical protocol to detect the intracellular localization of mRNAs in mesophyll cells of Nicotiana benthamiana (N. benthamiana). This protocol is based on poly (A) in situ hybridization method using an oligo d(T) probe conjugated with Alexa Fluor-488.
Keywords: poly (A) in situ hybridization, Nicotiana benthamiana, Virus-induced gene silencing, nucleoporin, mRNAs

[Abstract]

Materials and Reagents

  1. Leaves of 3-4 weeks old wild type or Nup gene silenced N. benthamiana
    Notes:
    a. This method can be applied for other dicot plants with relatively soft leaves.
    b. For Virus-induced gene silencing (VIGS) of N. benthamiana. See Zhang and Liu (2014).
  2. 10 µM 48-mer oligo d(T) 5’-labeled with Alexa Fluor-488, HPLC-purified [purchased from custom oligo services (e.g. Eurofins Genomics)]
    Note: Dissolved in TE (Tris-EDTA) buffer and store at -80 °C, shaded.
  3. 99.8% Methanol (4 ml per sample) (Wako pure chemical, special glade, catalog number: 131-01826 )
  4. 99.5% Ethanol (5 ml per sampl) (Wako pure chemical, special glade, catalog number: 057-00456 )
  5. 99.5% Ethanol/Xylene (1:1 v/v, 1 ml per sample) (Wako pure chemical, special glade, catalog number: 244-00086 )
  6. 99.8%Methanol/Fixation solutionB (1:1 v/v, 1 ml per sample)
  7. PerfectHyb Plus Hybridization Buffer (2 ml per sample) (Sigma-Aldrich, catalog number: H7033 )
  8. Fixation cocktail (see Recipes)
  9. Fixation solution A (see Recipes)
  10. Fixation solution B (see Recipes)

Equipment

  1. 3 ml vial containers
  2. Rotary shaker (e.g. BioCraft, model: BC-730 )
  3. Hybridization oven or incubator (50 °C) with rotary shaker (e.g. TAITEC, model: BR-23FP MR )
  4. Confocal laser-scanning microscope (preferred) or ordinary fluorescent microscope with the filter set for GFP or Alexa Fluor-488 (e.g. Olympus, model: FV1000-D )

Procedure

  1. Poly (A) RNA in situ hybridization
    1. Cut leaves of N. benthamiana into small pieces (e.g. 3 mm x 3 mm) by knife and put ~10 leaf pieces/sample in 3 ml vial container.
      Note that the center of larger pieces won’t be stained with AF488 probe.
    2. Add 1 ml Fixation solution A in the vial container and shake (Approx. 80 rpm) for 30 min at room temperature (RT). Leaf pieces will float on the surface of the solution at the start, but will sink later (Figure 1).

     
    Figure 1. Leaf discs of N. benthamiana in Fixation solution A before (left) and after (right) shaking for 30 min (step A2)

    1. Remove Fixation solution A by pipeting.
    2. Add 1 ml methanol. Shake (80 rpm) for 5 min at RT and remove methanol (twice). Note that the inner side of the vial should be washed well with methanol.
    3. Add 1 ml ethanol, shake (80 rpm) for 5 min at RT and remove ethanol (three times).
    4. Add 1 ml ethanol/xylene (1:1). Shake (80 rpm) for 30 min at RT and remove the solution. Note that leaf tissues will become white during steps A4-6.
    5. Add 1 ml 100% ethanol. Shake (80 rpm) for 5 min at RT and remove ethanol (twice).
    6. Add 1 ml 100% methanol. Shake (80 rpm) for 5 min at RT and remove ethanol (twice).
    7. Add methanol/Fixation solution B (1:1). Shake (80 rpm) for 5 min at RT and remove the solution.
    8. Add 1 ml Fixation solution A and shake (80 rpm) for 30 min at RT.
    9. Remove Fixation solution A.
    10. Add 1 ml Fixation solution B. Shake (80 rpm) for 5 min at RT and remove the solution (twice).
    11. Add 1 ml PerfectHyb Plus, and shake (80 rpm) for 5 min at RT.
    12.  Remove the solution and add 1 ml of fresh PerfectHyb Plus. Shake (80 rpm) at 50 °C in hybridization oven (incubator with rotary shaker can be used) for more than 1 h.
    13. Add 1 µl of 10 µM Alexa Fluor-488-labeled oligo d(T). Shake (80 rpm) at 50 °C overnight. It is preferred to perform fluorescent microscopy immediately after the preparation of samples, but the samples can be kept at least for a couple of days at RT, shaded.

  2. Microscopic observation
    1. Mount the sample in PerfectHyb Plus on slide glass with cover slip.
    2. Observe the localization of mRNA with confocal laser-scanning microscopy. Use appropriate setting for the detection of Alexa Fluor-488 (excitation peak 490 nm, emission peak 525 nm). We generally use 488-nm excitation source, and Alexa Fluor-488 fluorescence is recorded between 500 and 600 nm.


    Figure 2. Distribution of Poly (A) RNA in mesophyll cells of control (TRV-infected) or NbNup75-silenced Nicotiana benthamiana. In NbNup75-silenced plant, abnormal accumulation of mRNA in nuclei is observed as a result of impaired export of mRNA. Bars=30 µm. (Ohtsu et al. 2014)

Recipes

  1. Fixation cocktail (x 4)
    240 mM NaCl
    14 mM Na2HPO4
    6 mM NaH2PO4
    5.4 mM KCl
    160 mM EGTA
    Autoclaved and stored at RT
  2. Fixation solution A (for 10 ml)
    Fixation cocktail (x 4) 2.5 ml
    Formaldehyde 250 µl
    DMSO 500 µl
    Tween20 10 µl
    Heptan 5 ml
    H2O 1.74 ml
    Prepare before use
  3. Fixation solution B (for 10 ml)
    Fixation cocktail (x 4) 2.5 ml
    DMSO 500 µl
    Tween 20 10 µl
    Heptan 5 ml
    H2O 1.99 ml
    Prepare before use

Acknowledgments

This protocol was adapted from Parry et al. (2006) and Germain et al. (2010). The work was supported by a Grant-in-Aid for Scientific Research (B) (26292024) from the Japan Society for the Promotion of Science and by Grant for Basic Science Research Projects from the Sumitomo Foundation.

References

  1. Germain, H., Qu, N., Cheng, Y. T., Lee, E., Huang, Y., Dong, O. X., Gannon, P., Huang, S., Ding, P., Li, Y., Sack, F., Zhang, Y. and Li, X. (2010). MOS11: a new component in the mRNA export pathway. PLoS Genet 6(12): e1001250.
  2. Ohtsu, M., Shibata, Y., Ojika, M., Tamura, K., Hara-Nishimura, I., Mori, H., Kawakita, K. and Takemoto, D. (2014). Nucleoporin 75 is involved in the ethylene-mediated production of phytoalexin for the resistance of Nicotiana benthamiana to Phytophthora infestans. Mol Plant Microbe Interact 27(12): 1318-1330.
  3. Parry, G., Ward, S., Cernac, A., Dharmasiri, S. and Estelle, M. (2006). The Arabidopsis SUPPRESSOR OF AUXIN RESISTANCE proteins are nucleoporins with an important role in hormone signaling and development. Plant Cell 18(7): 1590-1603.
  4. Zhang, H. and Liu, Y. (2014). VIGS assays. Bio-protocol 4(5): e1057.

材料和试剂

  1. 3-4周龄野生型或Nup 基因沉默的叶子。 本bentiana
    注意:
    a。 该方法可应用于其他具有相对软叶的双子叶植物。
    b。 对于本塞姆氏烟草的病毒诱导的基因沉默(VIGS)。 见张和刘(2014)。
  2. 用HPLC纯化的Alexa Fluor-488(购自定制寡核苷酸服务(例如Eurofins Genomics))对10μM48聚体寡聚d(T)进行5'-标记]
    注意:溶于TE(Tris-EDTA)缓冲液并储存于-80℃,阴影处。
  3. 99.8%甲醇(每个样品4ml)(Wako pure chemical,special glade,目录号:131-01826)
  4. 99.5%乙醇(每个样品5ml)(Wako pure chemical,special glade,目录号:057-00456)
  5. 99.5%乙醇/二甲苯(1:1v/v,每个样品1ml)(Wako纯化学品,特殊空间,目录号:244-00086)
  6. 99.8%甲醇/固定溶液B(1:1v/v,每个样品1ml)
  7. PerfectHyb Plus杂交缓冲液(每个样品2ml)(Sigma-Aldrich,目录号:H7033)
  8. 固定鸡尾酒(见配方)
  9. 固定解决方案A(参见配方)
  10. 固定溶液B(参见配方)

设备

  1. 3 ml小瓶容器
  2. 旋转振荡器(例如 BioCraft,型号:BC-730)
  3. 具有旋转振荡器(例如TAITEC,型号:BR-23FP MR)的杂交炉或培养箱(50℃)
  4. 具有用于GFP或Alexa Fluor-488(例如Olympus,型号:FV1000-D)的滤光片的共聚焦激光扫描显微镜(优选)或普通荧光显微镜,

程序

  1. Poly(A)RNA原位杂交
    1. 剪切叶子的N. 本哈姆纳成小块(例如 3 mm x 3 mm) 刀并放入3ml小瓶容器中〜10片叶/样品。
      请注意,较大的中心不会被AF488探头染污。
    2. 在小瓶容器中加入1ml固定溶液A,摇动 80 rpm)在室温(RT)下30分钟。 叶片将浮在上面 表面的溶液在开始,但会后来下沉(图 1)。

     
    图1. N的叶片。 固定溶液A前(左)和后(右)摇动30分钟(步骤A2)之后

    1. 通过移液除去固定溶液A.
    2. 加入1ml甲醇。 在室温下摇动(80rpm)5分钟,除去甲醇(两次)。 注意 小瓶的内侧应用甲醇充分洗涤
    3. 加入1ml乙醇,在室温下摇动(80rpm)5分钟,除去乙醇(三次)
    4. 加入1ml乙醇/二甲苯(1:1)。 在室温下摇动(80rpm)30分钟 删除解决方案。 注意叶组织在白色会变白 步骤A4-6。
    5. 加入1ml 100%乙醇。 在室温下摇动(80rpm)5分钟,除去乙醇(两次)
    6. 加入1ml 100%甲醇。 在室温下摇动(80rpm)5分钟,除去乙醇(两次)
    7. 加入甲醇/固定溶液B(1:1)。 在室温下摇动(80rpm)5分钟并除去溶液
    8. 加入1ml固定溶液A,在室温下摇动(80rpm)30分钟
    9. 删除固定解决方案A.
    10. 加入1ml固定溶液B.在室温下摇动(80rpm)5分钟,并除去溶液(两次)
    11. 加入1ml PerfectHyb Plus,在室温下摇动(80rpm)5分钟
    12.  取出溶液,加入1 ml新鲜PerfectHyb Plus。 摇(80 rpm)在50℃下在杂交炉(具有旋转振荡器的培养箱中)   使用)1小时以上。
    13. 加入1μl的10μMAlexa Fluor-488标记的寡聚d(T)。 在50℃下摇动(80rpm)过夜。 它是 优选在紧接着之后进行荧光显微镜 样品的制备,但样品可以保持至少a 几天在RT,阴影

  2. 显微镜观察
    1. 将样品装在PerfectHyb Plus上的带有盖玻片的载玻片上。
    2. 用共焦激光扫描观察mRNA的定位 显微镜。 使用适当的设置检测Alexa Fluor-488   (激发峰490nm,发射峰525nm)。 我们一般使用488-nm   激发源,并记录Alexa Fluor-488荧光   500和600nm。


    图2.在对照(TRV感染的)或NbNup75 - 沉默的本氏烟草的叶肉细胞中Poly(A)RNA的分布。 em> NbNup75 - 沉默的植物,观察到核中mRNA的异常积累是mRNA输出受损的结果。 条=30μm。 (Ohtsu 2014)

食谱

  1. 固定鸡尾酒(×4)
    240mM NaCl 14mM Na 2 HPO 4
    6mM NaH 2 PO 4 sub/vbs 5.4 mM KCl
    160 mM EGTA
    自动保存并在RT保存
  2. 固定溶液A(10ml)
    固定鸡尾酒(×4)2.5 ml
    甲醛250μl
    DMSO500μl
    Tween20 10μl
    庚庚5毫升
    h H 2 O 1.74ml
    使用前准备
  3. 固定溶液B(10ml)
    固定鸡尾酒(×4)2.5 ml
    DMSO500μl
    吐温20 10微升
    庚庚5毫升
    H O 1.99ml
    使用前准备

致谢

该方案改编自Parry等人(2006)和Germain等人(2010)。 这项工作得到了日本科学促进会科学研究资助(B)(26292024)的支持。 和住友基金会的基础科学研究项目的Grant。

参考文献

  1. 本研究结果表明,该方法可以有效地抑制大肠杆菌感染的发生,但是, F.,Zhang,Y。和Li,X。(2010)。 MOS11:mRNA出口途径中的新组件 PLoS Genet 6(12):e1001250。
  2. Ohtsu,M.,Shibata,Y.,Ojika,M.,Tamura,K.,Hara-Nishimura,I.,Mori,H.,Kawakita,K.and Takemoto, Nucleoporin 75参与乙烯介导的植物抗毒素的生产,以抵抗本尼托克烟到Phytophthora infestans。 Mol Plant Microbe Interact 27(12):1318-1330。
  3. Parry,G.,Ward,S.,Cernac,A.,Dharmasiri,S。和Estelle,M。(2006)。 拟南芥 AUXIN RESISTANCE抑制剂蛋白质是核蛋白质,在其中具有重要作用激素信号传导和发育。 植物细胞 18(7):1590-1603
  4. Zhang,H.and Liu,Y。(2014)。 VIGS测定生物协议 4(5):e1057。
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How to cite this protocol: Mizuno, Y. and Takemoto, D. (2015). Detection of Poly (A) RNA in Mesophyll Cells of Nicotiana benthamiana Using in situ Hybridization. Bio-protocol 5(17): e1576. DOI: 10.21769/BioProtoc.1576; Full Text



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