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Northwestern Blot of Protein-RNA Interaction from Young Rice Panicles
RNA-蛋白质印记法研究水稻幼穗中蛋白质-RNA的相互作用   

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Abstract

The northwestern assay is employed to study the interaction between protein and RNA. The RNA binding proteins tend to bind to different kinds of RNA through either known domains or unknown sequences of proteins. Rice LGD1 recombinant protein, a grass-specific novel protein with RNA binding sequences in its C-terminal, was used to probe its function as an RNA binding protein. The LGD1 comprising von Willebrand factor type-A domain (vWA), coiled-coil and nuclear localization signal (NLS) is a class of protein that localizes both in the nucleus and cytoplasm. Although LGD1 does not contain any putative RNA binding domains, we could find high-affinity RNA binding residues at the C-terminus using ‘RNABindR’ prediction software (Terribilini et al., 2007). The LGD1 recombinant protein, purified from bacteria, somehow forms both dimer and monomer even under denaturing conditions. However, only the dimeric form is able to bind to total and mRNAs. Due to its reproducibility and reliability, we believe that this protocol can be used across different organisms.

Keywords: LGD1(lgd1), Multiple transcripts(多个转录), RNA binding(RNA结合), Alternative splicing(选择性剪接), Rice(水稻)

Materials and Reagents

  1. Young panicle tissues of wild type (Oryza sativa L. ssp. Japonica cultivar Tainung67) plants
  2. Tissues were collected from young panicles while they were in secondary branch initiation
  3. Recombinant GST-LGD1 protein from E. coli (BL21)
  4. pGEX-4T-1 GST expression vector (GE Healthcare, catalog number: 28-9545-49 )
  5. Total RNA isolation kit - Trizol (Life Technologies, InvitrogenTM, catalog number:  15596-026 )
  6. mRNA purification kit - Oligotex mRNA Mini Kit (QIAGEN, catalog number:  72022 )
  7. RNA 5'-end Labeling kit (T4 Polynucleotide Kinase) (Thermo Fisher Scientific, catalog number:  EK0031 )
  8. FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, catalog number:  EF0651 )
  9. [γ-32P]-ATP–for isotope labeling
  10. PVDF Immobilon-P membrane (EMD Millipore, catalog number:  IPVH15150 )
  11. G-50 gel filtration dye terminator removal column (Geneaid, Taiwan, catalog number: CG050 )
  12. Primary anti-GST antibody (Bioman, Taiwan, catalog number: GST001M ); HRP-conjugated secondary antibody (Thermo Fisher Scientific, catalog number: SA1-9510 )
  13. SuperSignal West Pico Chemiluminescence detection system (Thermo Fisher Scientific, catalog number: 34087 )
  14. Blocking buffer (see Recipes)

Equipment

  1. Gel electrophoresis (Bio-Rad Laboratories, catalog number: 165-8005EDU )
  2. SNAP i.d.® 2.0 protein detection system
  3. Equipment for phosphor imaging (Bio-Rad Laboratories, catalog number: 170-9460 )

Procedure

  1. RNA isolation and end-labeling of total and mRNA
    1. Total RNA was isolated from young panicles of wild type plants with Trizol kit.
    2. ~0.5 g tissue was used to isolate ~500 μg of total RNA and 200 μg total RNA was used to purify ~2 μg mRNA using Oligotex mRNA Mini Kit. Nanodrop was used for final RNA quantitation (~2 μg/μl for total RNA; ~20 ng/μl for mRNA).
    3. 5 μg total RNA and 1 μg mRNA were first treated with FastAP Thermosensitive Alkaline Phosphatase (10 U) at 37 °C for 1 h to remove 5’-phosphate group according to manufacturer’s protocol.
    4. The total and mRNAs were end-labeled with [γ-32P]-ATP using 1 μl (10 U) T4 Polynucleotide kinase at 37 °C for 30 min.
    5. Labeled RNAs were separated from unincorporated label by gel filtration on Sephadex G-50 column.
  2. Northwestern blot assay
    1. GST-LGD1 fusion protein was extracted from E. coli  harboring pGEX-4T-1 GST expression vector according to manufacturer’s protocol.
    2. The proteins were separated on 12% SDS PAGE under denaturing conditions, and transferred to PVDF Immobilon-P membrane and detected using the SNAP i.d. protein detection system as follows.
    3. For the Western blot, the primary anti-GST antibody (1:2,000) and secondary HRP (horseradish peroxidase)-conjugated antibody (1:3,000) were used for the enhanced chemiluminescence (ECL) detection.
    4. SuperSignal West Pico Chemiluminescence was employed to detect western signals according to the manufacturer’s instructions.
    5. For the Northwestern, recombinant proteins separated on 12% SDS PAGE under denaturing conditions were transferred to PVDF Immobilon-P membrane.
    6. The proteins were then renatured on the blots overnight at 4 °C in a renaturation buffer containing 0.1 M Tris HCl (pH 7.5) and 0.1% (v/v) NP-40.
    7. Blots were washed 4 times 15 min each in renaturation buffer and incubated 5 min at room temperature in blocking buffer.
    8. Blots were hybridized overnight at 4 °C in blocking buffer (without BSA) in the presence of labeled total and mRNAs.
    9. Finally, the blots were washed 4 times 5 min each in blocking buffer (without BSA and Triton) and autoradiographed to obtain the signal as seen in Figure 1.

Representative data

Figure 1. Northwestern blot analysis of LGD1 recombinant protein. The recombinant GST-LGD1 was purified from E. coli and analyzed through SDS-PAGE, western and northwestern blots. (a) Coomassie Brilliant Blue (CBB) staining shows two major bands (arrows). The size of the lower band is ~56 kD (monomer) and upper band is ~112 kD (homodimer). (b) Anti-GST antibody recognizes the GST-LGD1 fusion protein (arrows) as well as GST (open circle). Northwestern blot shows the total RNA. (c) and mRNA (d) isolated from young panicle tissues bind to upper band (arrows), but not to the lower band (open arrowhead). Asterisks indicate RNA binding to degraded form of LGD1.

Recipes

  1. Blocking buffer
    10 mM Tris HCl (pH 7.5)
    5 mM Mg acetate
    2 mM DTT
    5 % (w/v) BSA
    0.01 % (v/v) Triton X-100

Acknowledgments

We greatly appreciate the contributions of Drs Yue-Ie Hsing, Chyr-Guan Chern, Ming-Jen Fan and Su-May Yu to generate the TRIM database. We also thank Drs Ko Shimamoto (Nara Institute of Science and Technology, Japan), for sharing the pANDA-RNAi vector, and Su-May Yu (Institute of Molecular Biology, Academia Sinica), for excellent support with rice transgenic experiments. We also thank Ms Lin-Yun Kuang (Transgenic Plant Laboratory, Academia Sinica) for assistance in particle bombardment. We are grateful to Ms. Krisa Fredrickson for her English editing. This work was supported by research grants from Academia Sinica (Taiwan), the National Science and Technology Program for Agricultural Biotechnology (NSTP/AB, 098S0030055-AA, Taiwan), the National Science Council (98-2313-B-001-001-MY3, Taiwan) and the Li Foundation (USA) to Guang-Yuh, Jauh.

References

  1. Uyttewaal, M., Mireau, H., Rurek, M., Hammani, K., Arnal, N., Quadrado, M. and Giege, P. (2008). PPR336 is associated with polysomes in plant mitochondria. J Mol Biol 375(3): 626-636.
  2. Terribilini, M., Sander, J. D., Lee, J. H., Zaback, P., Jernigan, R. L., Honavar, V. and Dobbs, D. (2007). RNABindR: a server for analyzing and predicting RNA-binding sites in proteins. Nucleic Acids Res 35(Web Server issue): W578-584.
  3. Thangasamy, S., Chen, P. W., Lai, M. H., Chen, J. and Jauh, G. Y. (2012). Rice LGD1 containing RNA binding activity affects growth and development through alternative promoters. Plant J 71(2): 288-302.
  4. Uyttewaal, M., Mireau, H., Rurek, M., Hammani, K., Arnal, N., Quadrado, M. and Giege, P. (2008). PPR336 is associated with polysomes in plant mitochondria. J Mol Biol 375(3): 626-636.

简介

使用西北测定来研究蛋白质和RNA之间的相互作用。 RNA结合蛋白倾向于通过蛋白质的已知结构域或未知序列结合不同种类的RNA。将水稻LGD1重组蛋白(一种在其C末端具有RNA结合序列的草特异性新蛋白)用于探测其作为RNA结合蛋白的功能。包含von Willebrand因子A型结构域(vWA),卷曲螺旋和核定位信号(NLS)的LGD1是一类定位于细胞核和细胞质中的蛋白质。虽然LGD1不包含任何推定的RNA结合结构域,我们可以使用'RNABindR'预测软件在C末端找到高亲和力RNA结合残基(Terribilini等人,2007)。从细菌纯化的LGD1重组蛋白以某种方式甚至在变性条件下形成二聚体和单体。然而,仅二聚体形式能够结合总RNA和mRNA。由于其重现性和可靠性,我们认为该协议可以用于不同的生物。

关键字:lgd1, 多个转录, RNA结合, 选择性剪接, 水稻

材料和试剂

  1. 野生型( 0 sativa L. ssp 。粳稻栽培品种Tainung67)植物的青穗组织
  2. 当它们处于二次分支起始时,从年轻穗收集组织
  3. 来自E的重组GST-LGD1蛋白。 大肠杆菌(BL21)
  4. pGEX-4T-1GST表达载体(GE Healthcare,目录号:28-9545-49)
  5. 总RNA分离试剂盒 - Trizol(Life Technologies,Invitrogen TM ,目录号:15596-026)
  6. mRNA纯化试剂盒 - Oligotex mRNA Mini Kit(QIAGEN,目录号:72022)
  7. RNA 5'-末端标记试剂盒(T4多核苷酸激酶)(Thermo Fisher Scientific,目录号:EK0031)
  8. FastAP热敏碱性磷酸酶(Thermo Fisher Scientific,目录号:EF0651)
  9. [γ- 32 P] -ATP - 用于同位素标记
  10. PVDF Immobilon-P膜(EMD Millipore,目录号:IPVH15150)
  11. G-50凝胶过滤染料终止子去除柱(Geneaid,Taiwan,目录号:CG050)
  12. 原代抗GST抗体(Bioman,Taiwan,目录号:GST001M); HRP缀合的二抗(Thermo Fisher Scientific,目录号:SA1-9510)
  13. SuperSignal West Pico化学发光检测系统(Thermo Fisher Scientific,目录号:34087)
  14. 阻止缓冲区(参见配方)

设备

  1. 凝胶电泳(Bio-Rad Laboratories,目录号:165-8005EDU)
  2. SNAP i.d. ® 2.0蛋白检测系统
  3. 荧光成像设备(Bio-Rad Laboratories,目录号:170-9460)

程序

  1. RNA分离和总和mRNA的末端标记
    1. 使用Trizol试剂盒从野生型植物的幼穗中分离总RNA。
    2. 〜0.5g组织用于分离〜500μg总RNA,使用200μg总RNA用Oligotex mRNA Mini Kit纯化〜2μgmRNA。 使用Nanodrop进行最终RNA定量(对于总RNA约2μg/μl;对于mRNA约20ng /μl)。
    3. 首先用FastAP热敏碱性磷酸酶(10U)在37℃下处理5μg总RNA和1μgmRNA 1小时以根据制造商的方案除去5'-磷酸基团。
    4. 使用1μl(10U)T4多核苷酸激酶在37℃下用[γ-32 P] -ATP对总体和mRNA进行末端标记30分钟。
    5. 通过在Sephadex G-50柱上凝胶过滤从未结合的标记物中分离标记的RNA
  2. 西北印迹测定
    1. 从E中提取GST-LGD1融合蛋白。 大肠杆菌 含有pGEX-4T-1 GST表达载体
    2. 在变性条件下在12%SDS PAGE上分离蛋白质,并转移至PVDF Immobilon-P膜,并使用SNAP i.d. 蛋白检测系统如下。
    3. 对于Western印迹,使用一级抗GST抗体(1:2,000)和二级HRP(辣根过氧化物酶) - 缀合的抗体(1:3,000)用于增强的化学发光(ECL)检测。
    4. SuperSignal West Pico化学发光法用于根据制造商的说明书检测西方信号
    5. 对于西北部,在变性条件下在12%SDS PAGE上分离的重组蛋白转移到PVDF Immobilon-P膜。
    6. 然后将蛋白质在含有0.1M Tris HCl(pH7.5)和0.1%(v/v)NP-40的复性缓冲液中在4℃下在印迹上复性过夜。
    7. 印迹在复性缓冲液中洗涤4次,每次15分钟,并在室温下在封闭缓冲液中孵育5分钟。
    8. 在标记的总和mRNA存在下,印迹在4℃下在封闭缓冲液(无BSA)中杂交过夜。
    9. 最后,将印迹在封闭缓冲液(无BSA和Triton)中洗涤4次,每次5分钟,并放射自显影以获得如图1所示的信号。

代表数据

图1. LGD1重组蛋白的西北印迹分析 从 E中纯化重组GST-LGD1。并通过SDS-PAGE,western和westernwestern印迹分析。 (a)考马斯亮蓝(CBB)染色显示两个主要条带(箭头)。下带的大小为〜56kD(单体),上带为〜112kD(同二聚体)。 (b)抗GST抗体识别GST-LGD1融合蛋白(箭头)以及GST(空心圆圈)。西北印迹显示总RNA。 (c)和从年轻穗轴组织分离的mRNA(d)结合上带(箭头),但不结合下带(空心箭头)。星号表示RNA与LGD1的降解形式的结合。

食谱

  1. 阻塞缓冲区
    10mM Tris HCl(pH7.5) 5mM Mg醋酸盐 2 mM DTT
    5%(w/v)BSA 0.01%(v/v)Triton X-100

致谢

我们非常感谢岳兴兴博士,陈建文,明仁芬和苏玉宇创建TRIM数据库。我们还感谢Shimamoto博士(日本奈良科学技术研究所),共享pANDA-RNAi载体和Su-May Yu(中国科学院分子生物学研究所),为水稻转基因实验提供了极好的支持。我们还感谢中国科学院转基因植物实验室的协助粒子轰击。我们感谢Krisa Fredrickson女士的英语编辑。这项工作得到中央研究院(台湾),国家农业生物技术国家科技计划(NSTP/AB,098S0030055-AA,台湾),国家科学委员会(98-2313-B-001-001- MY3,台湾)和李基金会(美国)到光玉,Jauh。

参考文献

  1. Uyttewaal,M.,Mireau,H.,Rurek,M.,Hammani,K.,Arnal,N.,Quadrado,M.and Giege,P.(2008)。 PPR336与植物线粒体中的多核糖体相关。 J Mol Biol 375(3):626-636
  2. Terribilini,M.,Sander,J.D.,Lee,J.H.,Zaback,P.,Jernigan,R.L.,Honavar,V.and Dobbs,D。(2007)。 RNABindR:用于分析和预测蛋白质中RNA结合位点的服务器 Nucleic Acids Res 35(Web Server问题):W578-584
  3. Thangasamy,S.,Chen,P.W.,Lai,M.H.,Chen,J.and Jauh,G.Y.(2012)。 含有RNA结合活性的水稻LGD1通过替代启动子影响生长和发育。 Plant J 71(2):288-302。
  4. Uyttewaal,M.,Mireau,H.,Rurek,M.,Hammani,K.,Arnal,N.,Quadrado,M.and Giege,P.(2008)。 PPR336与植物线粒体中的多核糖体相关。 J Mol Biol em> 375(3):626-636。

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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Thangasamy, S. and Jauh, G. (2013). Northwestern Blot of Protein-RNA Interaction from Young Rice Panicles. Bio-protocol 3(7): e625. DOI: 10.21769/BioProtoc.625.
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