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Mar 2021
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Enrichment of Cytoplasmic RNA Granules from Arabidopsis Seedlings
从拟南芥幼苗中富集细胞质 RNA 颗粒   

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Abstract

RNA granules (RGs) are membraneless intracellular compartments that play important roles in the post-transcriptional control of gene expression. Stress granules (SGs) are a type of RGs that form under environmental challenges and/or internal cellular stresses. Stress treatments lead to strong mRNAs translational inhibition and storage in SGs until the normal growth conditions are restored. Intriguingly, we recently showed that plant stress granules are associated with siRNA bodies, where the RDR6-mediated and transposon-derived siRNA biogenesis occurs (Kim et al., 2021). This protocol provides a technical workflow for the enrichment of cytoplasmic RGs from Arabidopsis seedlings. We used the DNA methylation-deficient ddm1 mutant in our study, but the method can be applied to any other plant samples with strong RG formation. The resulting RG fractions can be further tested for either RNAs or proteins using RNA-seq and mass spectrometry-based proteomics.

Keywords: RNA granule (RNA颗粒 ), Stress granule (应激颗粒), siRNA body (siRNA 体), Transposon (转座子), DDM1 (DDM1)

Background

RNA granules (RGs) are non-membraneous cellular architectures that are relevant to a variety of biological processes. Of these, stress granules (SGs) contain non-translating mRNAs and various RNA-binding proteins, and serve as the assorting sites of mRNAs for storage, translational reinitiation, or degradation (Anderson and Kedersha, 2009). Recently, we demonstrated that plant SGs include numerous transposon RNAs in DNA methylation-deficient mutants (Kim et al., 2021). Being natural endogenous mutagens in genomes, transposons are counteracted by the host’s epigenetic silencing mechanisms, which are primarily mediated by siRNAs. Several studies have suggested that transposon-derived siRNAs are produced in the siRNA bodies, which are often in association with SGs (McCue et al., 2012 and 2013). The transcriptome of SGs in yeast and human have been characterized in detail (Jain et al., 2016; Khong et al., 2017), revealing that SG-located RNAs are depleted of ribosomes and relatively longer in length. Consistently, our latest work also showed for the first time in a plant system that SGs contain weakly translating RNAs, the majority of which are derived from transposons (Kim et al., 2021). Given the importance and prevalence of RGs in a wide variety of biological processes, the identification of their RNA and protein components is a critical first step towards understanding RG-mediated gene expression control. Therefore, we describe here a versatile method for the enrichment of the RGs from Arabidopsis seedlings.

Materials and Reagents

  1. Whatman filter paper (Merk, catalog number: WHA1001150)

  2. Pipette tip 1,000 μl (Axygen, catalog number: T-1000-C-L-R-S)

  3. Pipette tip 10 ml (Eppendorf, catalog number: 0030000781)

  4. 1.5 ml microcentrifuge tube (Axygen, catalog number: MCT-150-C-ZX)

  5. 50 ml centrifuge tube (Corning, catalog number: 430828)

  6. Petri dish (any brand, 47 mm diameter)

  7. Funnel (any brand, 100 mm diameter)

  8. Arabidopsis ddm1-2 mutant in the Columbia-0 background

  9. Ethanol (Merk, catalog number: 51976)

  10. Triton X-100 (Merk, catalog number: T8787)

  11. Murashige and Skoog basal medium with Vitamins (PhytoTech, catalog number: M519)

  12. Distilled water, generated using the RSJ Water Purification system (Tanon, catalog number: RODI-220B1)

  13. Liquid nitrogen

  14. Miracloth (Sigma-Aldrich, catalog number: 475855)

  15. Tris base (Fisher Scientific, catalog number: BP152-500)

  16. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473)

  17. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A466-250)

  18. Potassium acetate (KOAc) (Sigma-Aldrich, catalog number: P1190)

  19. Magnesium acetate (MgOAc) (Sigma-Aldrich, catalog number: 63052)

  20. Dithiothreitol (DTT) (Fisher Scientific, catalog number: R0861)

  21. Nonylphenyl-polyethylene Glycol (NP-40) (Fisher Scientific, catalog number: 49-201)

  22. cOmpleteTM, EDTA-free protease inhibitor cocktail (Sigma-Aldrich, catalog number: 11873580001)

  23. RNasin® Plus RNase inhibitor (Promega, catalog number: N2615)

  24. Sterilization solution (see Recipes)

  25. Half-strength MS-medium plate (see Recipes)

  26. RG lysis buffer (see Recipes)

Equipment

  1. Pipette 1,000 μl, 10 ml (Eppendorf, catalog numbers: 3123000063, 4720000011)

  2. Vortexer (any brand)

  3. Clean bench (any brand)

  4. Refrigerator (4°C) (any brand)

  5. Plant growth chamber (any model with temperature and light control)

  6. Mortar and pestle (any brand)

  7. Sorvall LYNX 4000 Superspeed Centrifuge (ThermoFisher, catalog number: 75006580)

  8. Microcentrifuge 5424R (Eppendorf, model: 5424R)

  9. Centrifuge 5810R (Eppendorf, model: 5810R, catalog number: 022625101)

Procedure

  1. Overview

    This protocol allows for the simple and fast enrichment of RG fractions (albeit crude) from plant samples. Briefly, the RG fractions are separated and dissolved by continuous centrifugation with RG lysis buffer. The enriched RG fractions can be subsequently subjected to RNA-seq and protein analysis (Figure 1).



    Figure 1. Scheme of RNA granule enrichment


  2. Plant preparation

    1. Place 100 μl of ddm1-2 seeds in a 1.5 ml microcentrifuge tube.

    2. Add 1 ml of sterilization solution to seeds and vortex for 4 min (see Recipe 1).

    3. Discard the solution.

    4. Wash the seed with 1 ml of 100% ethanol.

    5. Vortex for 1 min and discard the ethanol.

    6. Repeat Steps B4 and B5.

    7. Prepare a sheet of filter paper and pipet the seeds onto the paper.

    8. Let the ethanol evaporate for 3 min.

    9. Pick up the paper and sprinkle the seeds onto half-strength Murashige and Skoog medium (see Recipe 2).

    10. Wrap the plate with parafilm and place it into a 4°C chamber for 2 days.

    11. Transfer the plate into a growth chamber set at 22°C and under 16 h light/8 h dark cycles for 10 days.

    Notes:

    1. Any plant samples in addition to Arabidopsis seedlings can be used for this protocol.

    2. Perform seed sterilization on a clean bench (wipe down with 70% ethanol before use).


  3. RNA granule enrichment

    1. Grind 2 g of seedlings into a fine powder in liquid nitrogen using a precooled mortar and pestle.

    2. Collect the samples (approximately 5 ml) into a 50 ml tube and resuspend in 5 ml of RG lysis buffer (see Recipe 3).

    3. Filter the resulting slurry through four layers of Miracloth in a funnel to a 50 ml conical tube and centrifuge at 850 × g for 5 min at 4°C to pellet cell debris.

    4. Transfer the supernatant to a new 50 ml tube and add 5 ml of RG lysis buffer.

    5. Centrifuge at 4,000 × g for 10 min at 4°C and discard the supernatant.

    6. Resuspend the pellet in 2 ml of RG lysis buffer. Centrifuge at 18,000 × g for 10 min at 4°C.

    7. Resuspend the pellet in 2 ml of RG lysis buffer, vortex, and centrifuge at 18,000 × g at 4°C for 10 min.

    8. Discard the supernatant and resuspend the pellets gently in 1 ml of RG lysis buffer. Centrifuge at 850 × g for 10 min at 4°C.

    9. Transfer the supernatant (enriched with RGs) into a 1.5 ml microcentrifuge tube without disturbing any residue and keep it in a freezer until use.

    Note: We recommend using the fluorescence-tagged RG-marker plant lines to quickly check for successful RG enrichment.


  4. RNA analysis

    The final RG fraction resulting from the protocol described above can be subjected to regular RNA extraction and subsequently tested for either targeted RNA analyses using RT-qPCR or transcriptome-wide profiling with RNA-seq.

    Note: Refer to Kim et al. (2021) for suggestions on any RG-specific marker genes.

Data analysis

RNA-seq data generated from the RG enrichment fractions can be analyzed as detailed in the original paper (Kim et al., 2021; https://doi.org/10.1038/s41477-021-00867-4).

Recipes

  1. Sterilization solution

    70% ethanol

    0.05% Triton X-100

  2. Half-strength MS-medium plate

    2.2 g/L Murashige and Skoog basal medium with Vitamins

    Adjust pH to 5.7 with KOH

    7 g/L plant agar

    Sterilize by autoclaving

  3. RG lysis buffer

    50 mM Tris-HCl, pH 7.4

    100 mM KOAc

    2 mM MgOAc

    0.5% NP-40

    0.5 mM DTT

    One tablet (in 50 ml) of protease inhibitor cocktail (cOmpleteTM, EDTA-free Protease Inhibitor Cocktail)

    1 U/μl RNasin Plus RNase Inhibitor


    1 M Tris-HCl, pH 7.4 2.5 ml
    1 M KOAc 5 ml
    1 M MgOAc 0.1 ml
    10% NP-40 2.5 ml
    1 M DTT 25 μl
    Protease inhibitor cocktail One tablet
    40,000 U/ml RNasin Plus RNase Inhibitor 1.25 ml
    Distilled water Top up to 50 ml


Notes:

  1. Prepare all solutions and buffers with distilled water.

  2. Add DTT and RNase Inhibitor right before use.

Acknowledgments

The work was supported by grants from the National Natural Science Foundation of China (31970518), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB27030209), and the General Program of Natural Science Foundation of Shanghai (21ZR1470700). E. Y. Kim is the recipient of a President’s International Fellowship Initiative (PIFI) young staff fellowship (2021FYB0001) from CAS. This protocol was adapted from our previously reported work (Kim et al., 2021).

Competing interests

The authors declare no conflicts of interest.

References

  1. Anderson, P. and Kedersha, N. (2009). RNA granules: post-transcriptional and epigenetic modulators of gene expression. Nat Rev Mol Cell Biol 10(6): 430-436.
  2. Jain, S., Wheeler, J. R., Walters, R. W., Agrawal, A., Barsic, A. and Parker, R. (2016). ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164(3): 487-498.
  3. Khong, A., Matheny, T., Jain, S., Mitchell, S. F., Wheeler, J. R. and Parker, R. (2017). The stress granule transcriptome reveals principles of mRNA accumulation in stress granules. Mol Cell 68(4): 808-820e805.
  4. Kim, E. Y., Wang, L., Lei, Z., Li, H., Fan, W. and Cho, J. (2021). Ribosome stalling and SGS3 phase separation prime the epigenetic silencing of transposons. Nat Plants 7(3): 303-309.
  5. McCue, A. D., Nuthikattu, S., Reeder, S. H. and Slotkin, R. K. (2012). Gene expression and stress response mediated by the epigenetic regulation of a transposable element small RNA. PLoS Genet 8(2): e1002474.
  6. McCue, A. D., Nuthikattu, S. and Slotkin, R. K. (2013). Genome-wide identification of genes regulated in trans by transposable element small interfering RNAs. RNA Biol 10(8): 1379-95.

简介

[摘要] RNA 颗粒 (RGs) 是无膜细胞内隔室,在基因表达的转录后控制中起重要作用。应力颗粒 (SG) 是一种在环境挑战和/或内部细胞压力下形成的 RG。压力处理导致强烈的 mRNA 翻译抑制和储存在 SGs 中,直到恢复正常的生长条件。有趣的是,我们最近发现植物应激颗粒与 siRNA 体相关,其中发生 RDR6 介导和转座子衍生的 siRNA 生物发生(Kim等,2021)。该协议为从拟南芥幼苗中富集细胞质 RGs 提供了技术工作流程。我们使用了 DNA 甲基化缺陷的ddm1突变体在我们的研究中,但该方法可以应用于任何其他具有强 RG 形成的植物样品。可以使用RNA 序列和基于质谱的蛋白质组学对得到的 RG 级分进一步测试 RNA 或蛋白质。


[背景] RNA 颗粒 (RG) 是与各种生物过程相关的非膜细胞结构。其中,应激颗粒 (SG) 包含非翻译 mRNA 和各种 RNA 结合蛋白,并作为 mRNA 的分类位点用于储存、翻译重新启动或降解(Anderson 和 Kedersha,2009)。最近,我们证明植物 SG 在 DNA 甲基化缺陷突变体中包含大量转座子 RNA(Kim等,2021)。作为基因组中的天然内源性诱变剂,转座子被宿主的表观遗传沉默机制抵消,这些机制主要由 siRNA 介导。几项研究表明,转座子衍生的 siRNA 是在 siRNA 体中产生的,这通常与 SG 相关(McCue等,2012 和 2013)。酵母和人类 SGs 的转录组已被详细表征(Jain等人,2016 年;Khong等人,2017 年),揭示了 SG 定位的 RNA 缺乏核糖体并且长度相对较长。一致地,我们的最新工作也首次在植物系统中显示 SG 包含弱翻译 RNA,其中大部分来自转座子(Kim等,2021)。鉴于 RG 在各种生物过程中的重要性和普遍性,鉴定其 RNA 和蛋白质成分是了解 RG 介导的基因表达控制的关键第一步。因此,我们在这里描述了一种从拟南芥幼苗中富集 RGs 的通用方法。

关键字:RNA颗粒 , 应激颗粒, siRNA 体, 转座子, DDM1


材料和试剂

 
Whatman滤纸(Merk,目录号:WHA1001150)
移液器吸头 1,000 μl(Axygen,目录号:T-1000-CLRS)
移液器吸头 10 ml(Eppendorf,目录号:0030000781)
1.5 ml 微量离心管(Axygen,目录号:MCT-150-C-ZX)
50 ml离心管(Corning,目录号:430828)
培养皿(任何品牌,直径 47 毫米)
漏斗(任何品牌,直径 100 毫米)
Columbia-0 背景下的拟南芥ddm1-2突变体
乙醇(Merk,目录号:51976)
Triton X-100(Merk,目录号:T8787)
含有维生素的 Murashige 和 Skoog 基础培养基(PhytoTech,目录号:M519)
蒸馏水,使用 RSJ 水净化系统(Tanon,目录号:RODI-220B1)产生
液氮
Miracloth(Sigma-Aldrich,目录号:475855)
Tris 碱(Fisher Scientific,目录号:BP152-500)
氢氧化钾(KOH)(Sigma-Aldrich,目录号:221473)
盐酸(HCl)(Fisher Scientific,目录号:A466-250)
醋酸钾(KOAc)(Sigma-Aldrich,目录号:P1190)
醋酸镁(MgOAc)(Sigma-Aldrich,目录号:63052)
二硫苏糖醇(DTT)(Fisher Scientific,目录号:R0861)
壬基苯基聚乙二醇(NP-40)(Fisher Scientific,目录号:49-201)
cOmplete TM ,不含 EDTA 的蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:11873580001)
的RNasin ®加RNase抑制剂(Promega公司,目录号:N2615)
灭菌溶液(见配方)
半强度 MS 中板(见配方)
RG 裂解缓冲液(参见配方)
 
设备
 
吸管1000 μ升,将10毫升(的Eppendorf,产品目录号:3123000063,4720000011 )
涡流器(任何品牌)
超净工作台(任何品牌)
冰箱(4°C)(任何品牌)
植物生长室(任何具有温度和光控制的型号)
研钵和研杵(任何品牌)
Sorvall LYNX 4000 Superspeed Centrifuge(ThermoFisher,目录号:75006580 )
微量离心机 5424R(Eppendorf,型号:5424R)
离心机 5810R(Eppendorf,型号:5810R ,目录号:022625101 )
 
程序电子
 
概述
该协议允许从植物样品中简单快速地富集 RG 组分(尽管是原油)。简而言之,通过用 RG 裂解缓冲液连续离心来分离和溶解 RG 级分。随后可以对富集的 RG 分数进行 RNA 序列和蛋白质分析 (图 1)。
 
 
图 1. RNA 颗粒富集方案
 
植物准备
将 100 μl ddm1-2种子放入 1.5 ml 微量离心管中。
向种子中加入 1 ml 灭菌溶液并涡旋 4 分钟(参见配方 1)。
丢弃解决方案。
用 1 ml 100% 乙醇清洗种子。
涡旋 1 分钟并丢弃乙醇。
重复步骤 B4 和 B5。
准备一张滤纸并将种子移到纸上。
让乙醇蒸发 3 分钟。
拿起纸,将种子撒在半强度的 Murashige 和 Skoog 培养基上(参见配方 2)。
用封口膜包裹板,然后将其放入 4°C 的腔室中 2 天。
将板转移到 22°C 和 16 小时光照/8 小时黑暗循环下的生长室中,持续 10 天。
笔记:
除拟南芥幼苗外的任何植物样品均可用于该协议。
在干净的工作台上进行种子灭菌(使用前用 70% 乙醇擦拭)。
 
RNA颗粒富集
使用预冷的研钵和研杵将 2 克幼苗在液氮中研磨成细粉。
将样品(约 5 ml)收集到 50 ml 试管中并重悬在 5 ml RG 裂解缓冲液中(参见配方 3)。
将所得浆液通过漏斗中的四层 Miracloth 过滤到 50 ml 锥形管中,并在 4°C 下以850 × g离心5 分钟以沉淀细胞碎片。
将上清液转移到新的 50 ml 管中并加入 5 ml RG 裂解缓冲液。
在 4°C 下以 4,000 × g离心10 分钟并丢弃上清液。
将沉淀重悬在 2 ml RG 裂解缓冲液中。在 4°C 下以 18,000 × g离心10 分钟。
将沉淀重悬在 2 ml RG 裂解缓冲液中,涡旋并在 4°C 下以 18,000 × g离心10 分钟。
弃去上清液,用 1 ml RG 裂解缓冲液轻轻地重悬沉淀。在 4°C 下以 850 × g离心10 分钟。
将上清液(富含 RGs)转移到 1.5 ml 微量离心管中,不要干扰任何残留物,并将其保存在冰箱中直至使用。
注意:我们建议使用荧光标记的 RG 标记植物系来快速检查成功的 RG 富集。
 
RNA分析
由上述协议产生的最终 RG 部分可以进行常规 RNA 提取,随后使用 RT-qPCR 或转录组范围的分析进行靶向 RNA 分析测试。
注意:请参阅 Kim 等人。(2021)关于任何 RG 特异性标记基因的建议。
 
数据分析
 
可以按照原始论文(Kim等,2021;https://doi.org/10.1038/s41477-021-00867-4)中的详细说明分析从 RG 富集部分生成的 RNA-seq 数据。
 
食谱
 
杀菌溶液
70%乙醇
0.05% 海卫 X-100
半强度MS-中板
2.2 g/L Murashige 和 Skoog 基础培养基,含维生素
用 KOH 将 pH 值调节至 5.7
7 g/L 植物琼脂
高压灭菌
RG裂解缓冲液
50 mM Tris-HCl,pH 7.4
100 mM KOAc
2 mM MgOAc
0.5% NP-40
0.5 毫米 DTT
一片(50 毫升)蛋白酶抑制剂混合物(cOmplete TM ,不含 EDTA的蛋白酶抑制剂混合物)
1 U/μl RNasin Plus RNase Inhibitor
 
1 M Tris-HCl,pH 7.4
2.5 毫升
1 M KOAc
5毫升
1 M MgOAc
0.1毫升
10% NP-40
2.5 毫升
1 M 数字电视
25 微升
蛋白酶抑制剂鸡尾酒
一粒
40,000 U/ml RNasin Plus RNase Inhibitor
1.25 毫升
蒸馏水
最多 50 毫升
 
笔记:
用蒸馏水准备所有溶液和缓冲液。
使用前添加 DTT 和 RNase Inhibitor。
 
致谢
 
该工作得到了国家自然科学基金(31970518)、中国科学院战略性优先研究计划(XDB27030209)和上海市自然科学基金面上项目(21ZR1470700)的资助。EY Kim 是 CAS 总统国际奖学金计划 (PIFI) 青年员工奖学金 (2021FYB0001) 的获得者。该协议改编自我们之前报告的工作(Kim等人,2021 年)。
 
利益争夺
 
作者宣称没有利益冲突。
 
参考
 
Anderson, P. 和 Kedersha, N. (2009)。RNA 颗粒:基因表达的转录后和表观遗传调节剂。Nat Rev Mol Cell Biol 10(6): 430-436。
Jain, S.、Wheeler, JR、Walters, RW、Agrawal, A.、Barsic, A. 和 Parker, R.(2016 年)。ATPase 调节的应激颗粒包含多种蛋白质组和亚结构。单元格164(3):487-498。
Khong, A., Matheny, T., Jain, S., Mitchell, SF, Wheeler, JR 和 Parker, R. (2017)。应激颗粒转录组揭示了应激颗粒中 mRNA 积累的原理。 摩尔细胞68(4):808-820 e805。
Kim, EY, Wang, L., Lei, Z., Li, H., Fan, W. 和 Cho, J. (2021)。核糖体停滞和 SGS3 相分离引发转座子的表观遗传沉默。天然植物7(3):303-309。
McCue, AD, Nuthikattu, S., Reeder, SH 和 Slotkin, RK (2012)。由转座因子小 RNA 的表观遗传调控介导的基因表达和应激反应。PLoS 基因8(2):e1002474。
McCue, AD, Nuthikattu, S. 和 Slotkin, RK (2013)。全基因组鉴定通过转座因子小干扰 RNA 进行反式调节的基因。 RNA 生物学10(8):1379-95。
 
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引用:Lei, Z., Kim, E. Y. and Cho, J. (2021). Enrichment of Cytoplasmic RNA Granules from Arabidopsis Seedlings. Bio-protocol 11(21): e4212. DOI: 10.21769/BioProtoc.4212.
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