从拟南芥幼苗中富集细胞质 RNA 颗粒

Zhen  Lei; Eun Yu  Kim; Jungnam  Cho

doi:10.21769/BioProtoc.4212

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Peer-reviewed

Enrichment of Cytoplasmic RNA Granules from Arabidopsis Seedlings

从拟南芥幼苗中富集细胞质 RNA 颗粒

ZL Zhen Lei

EK Eun Yu Kim

JC Jungnam Cho email

发布: 2021年11月05日第11卷第21期 DOI: 10.21769/BioProtoc.4212 浏览次数: 2551

评审: Wenrong HeYuan WangYe Xu

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Cover of Nature Plants, featuring study using the protocol.

Mar 2021

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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

Whatman filter paper (Merk, catalog number: WHA1001150)
Pipette tip 1,000 μl (Axygen, catalog number: T-1000-C-L-R-S)
Pipette tip 10 ml (Eppendorf, catalog number: 0030000781)
1.5 ml microcentrifuge tube (Axygen, catalog number: MCT-150-C-ZX)
50 ml centrifuge tube (Corning, catalog number: 430828)
Petri dish (any brand, 47 mm diameter)
Funnel (any brand, 100 mm diameter)
Arabidopsis ddm1-2 mutant in the Columbia-0 background
Ethanol (Merk, catalog number: 51976)
Triton X-100 (Merk, catalog number: T8787)
Murashige and Skoog basal medium with Vitamins (PhytoTech, catalog number: M519)
Distilled water, generated using the RSJ Water Purification system (Tanon, catalog number: RODI-220B1)
Liquid nitrogen
Miracloth (Sigma-Aldrich, catalog number: 475855)
Tris base (Fisher Scientific, catalog number: BP152-500)
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473)
Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A466-250)
Potassium acetate (KOAc) (Sigma-Aldrich, catalog number: P1190)
Magnesium acetate (MgOAc) (Sigma-Aldrich, catalog number: 63052)
Dithiothreitol (DTT) (Fisher Scientific, catalog number: R0861)
Nonylphenyl-polyethylene Glycol (NP-40) (Fisher Scientific, catalog number: 49-201)
cOmplete^TM, EDTA-free protease inhibitor cocktail (Sigma-Aldrich, catalog number: 11873580001)
RNasin^® Plus RNase inhibitor (Promega, catalog number: N2615)
Sterilization solution (see Recipes)
Half-strength MS-medium plate (see Recipes)
RG lysis buffer (see Recipes)

Equipment

Pipette 1,000 μl, 10 ml (Eppendorf, catalog numbers: 3123000063, 4720000011)
Vortexer (any brand)
Clean bench (any brand)
Refrigerator (4°C) (any brand)
Plant growth chamber (any model with temperature and light control)
Mortar and pestle (any brand)
Sorvall LYNX 4000 Superspeed Centrifuge (ThermoFisher, catalog number: 75006580)
Microcentrifuge 5424R (Eppendorf, model: 5424R)
Centrifuge 5810R (Eppendorf, model: 5810R, catalog number: 022625101)

Procedure

English

中文翻译

文章信息

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如何引用

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.