利用小鼠组织总RNA的离体检测IRE1α依赖性RNA切割的实验方案

Mohammad Mamun Ur Rashid; So-Young Rah; Se-Woong Ko; Han-Jung Chae

doi:10.21769/BioProtoc.5414

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Peer-reviewed

An Ex Vivo Protocol to Assess IRE1α-Dependent RNA Cleavage Using Total RNA Isolated from Mouse Tissues

利用小鼠组织总RNA的离体检测IRE1α依赖性RNA切割的实验方案

MR Mohammad Mamun Ur Rashid ^{*, §a}

SR So-Young Rah ^{*, §b}

SK Se-Woong Ko

HC Han-Jung Chae email

(*Contributed equally to this work, §aTechnical contact: mamun.phiiuc@gmail.com, §bTechnical contact: syrah76@jbnu.ac.kr) 发布: 2025年08月20日第15卷第16期 DOI: 10.21769/BioProtoc.5414 浏览次数: 1823

评审: Al Borhan BayazidNavnita DuttaAnonymous reviewer(s)

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Abstract

Regulated IRE1-dependent decay (RIDD) is a critical cellular mechanism mediated by the endoplasmic reticulum (ER) stress sensor IRE1α, which cleaves a variety of RNA targets to regulate ER homeostasis. Current in vitro assays to study IRE1α activity largely rely on synthetic or in vitro transcribed RNA substrates, which may not fully replicate the physiological complexities of native RNA molecules. Here, we present a comprehensive protocol to assess IRE1α-dependent RNA cleavage activity using total RNA isolated directly from mouse tissues. This protocol provides a step-by-step guide for tissue collection, RNA isolation, an ex vivo RIDD assay, cDNA synthesis, and subsequent RT-PCR analysis of target mRNA cleavage products. Key reagents include active IRE1α protein, the RIDD-specific inhibitor 4μ8C, and target-specific primers for RIDD-regulated genes such asBloc1s1 and Col6a1. Quantitative assessment is achieved using agarose gel electrophoresis and imaging software. This methodology enables the study of IRE1α's RNA cleavage activity under conditions that closely mimic in vivo environments, providing a more physiologically relevant approach to understanding the role of RIDD in cellular and tissue-specific contexts.

Key features

• Uses total RNA from mouse tissues instead of synthetic RNA to better reflect in vivo conditions.

• Includes RIDD-specific controls such as IRE1α inhibitor (4μ8C) and RNase A to confirm targeted RNA cleavage.

• Combines agarose gel electrophoresis and ImageJ quantification for both qualitative and statistical validation.

• Allows comparative studies of IRE1α activity across multiple mouse tissues in different biological contexts.

Keywords: IRE1α (IRE1α)

RIDD (RIDD)

RNA Cleavage (RNA切割)

Endoplasmic reticulum (内质网)

ER stress (内质网应激)

Ex vivo (离体实验)

Graphical overview

Ex vivo regulated IRE1-dependent decay (RIDD) assay workflow

Background

Regulated IRE1-dependent decay (RIDD) is a cellular mechanism that was first described inDrosophila melanogaster cells, where the endoplasmic reticulum (ER) stress sensor IRE1 was observed to induce the degradation of mRNAs encoding proteins that transit through the ER [1]. Subsequent studies in mammalian cells reported that IRE1α suppressed the expression not only of secretory pathway cargo proteins but also of ER-resident proteins that handle the folding and trafficking of cargo proteins [1].

Under high ER stress, IRE1α acquires endonucleolytic activity against a large array of RNA targets, first identified inD. melanogaster and termed RIDD, including ER-localized mRNAs and non-coding RNAs in mammals [2]. The RIDD pathway was later confirmed in mammalian cells, but it has yet to be fully determined in fungi [3].

The physiological significance of RIDD has been explored in various contexts. RIDD has been shown to regulate IgM and IgG2b expression in B cells, while IgG1 response relies on XBP-1 [4]. RIDD has also been implicated in curtailing immunoglobulin secretion from plasma cells and regulating autophagy in plants by degrading the mRNAs of several negative regulators of autophagy [5,6]. Additionally, RIDD has been suggested to play cytoprotective roles, such as contributing to feedback control on proinsulin expression in pancreatic beta-cells or protecting liver cells from acetaminophen toxicity [7].

Previous studies have established robust in vitro protocols to assess the RNA cleavage activity of IRE1α, predominantly utilizing synthetic RNA substrates [8,9]. These methods often rely on short, specifically designed RNA sequences or in vitro–transcribed RNAs mimicking XBP1 mRNA or validated RIDD targets. Such approaches, while valuable, allow precise kinetic measurements of IRE1α activity and have significantly contributed to understanding its RNase function. However, these methods may oversimplify the complex structural and contextual variations found in native RNA molecules within a cellular or tissue environment. This limitation underscores the need for methodologies that better represent the physiological conditions in which IRE1α operates.

While existing protocols effectively demonstrate IRE1α's RNA cleavage activity using synthetic or specific RNA substrates in vitro, these approaches may not fully capture the physiological context of RNA folding and complexity inherent in native tissues. To address this limitation, we propose a methodology utilizing total RNA isolated directly from various mouse tissues. This approach aims to provide a comprehensive understanding of IRE1α's substrate specificity and activity under conditions that closely mimic in vivo environments. By introducing this innovative perspective, our study seeks to expand the applicability of in vitro RNA cleavage assays, offering a versatile tool to unravel the intricate regulatory roles of IRE1α in diverse biological contexts.

Materials and reagents

Biological materials

1. Collected tissues from C57BL/6 mouse, e.g., kidney and liver

2. PCR primers (Table 1)

Table 1. List of primers

Gene	Primer	Accession no.	Usage
Bloc1s1	F: 5'-CAAGGAGCCTGCAGGAGAAGA-3' (Tm= 58.4 °C) R: 5'-CCAGGAGGGTGAAGTAAGAGG-3' (Tm = 58.2 °C)	NM_015740.3	RT-PCR, qRT-PCR
Col6a1	F: 5'-TAGCCGCGATGCAGAAGAG-3' (Tm= 59 °C) R: 5'-TTCCTCGCTCCCCCTCATA-3' (Tm = 58.4 °C)	NM_009933.5	RT-PCR, qRT-PCR
Beta Actin	F: 5'-AGGCCAACCGTGAAAAGATGACC-3' (Tm= 62.7 °C) R: 5'-ACCGCTCGTTGCCAATAGTGATGA-3' (Tm= 62.7 °C)	NM_007393.5	RT-PCR
Beta Actin	F: 5'-GGCTGTATTCCCCTCCATCG-3' (Tm= 59 °C R: 5'-CCAGTTGGTAACAATGCCATGT-3' (Tm= 58.5 °C)	NM_007393.5	qRT-PCR
Stock concentration: 100 µM. Source: Bioneer Corporation, Daejeon, Republic of Korea.

Reagents

A. RNA isolation from different mouse tissues

1. Tri-RNA reagent (Favorgen Biotech Corp., catalog number: FATRR 001) (store at 4 °C; shelf life: 12 months)

2. Chloroform (Sigma-Aldrich, catalog number: C2432) (store at room temperature)

3. Isopropanol (Sigma-Aldrich, catalog number: I9616) (store at room temperature)

4. Ethanol (Sigma-Aldrich, catalog number: E7023) (store at room temperature)

5. Ultrapure^TM DNase/RNase-free distilled water (Invitrogen, catalog number: 10977015) (store at room temperature)

6. Agilent RNA 6000 Nano kit (Agilent Technologies, catalog number: 5067-1511) (store according to the manufacturer's instructions)

7. 1× PBS (Enzynomics, catalog number: EBP008-1000)

B. RNA cleavage assay of isolated RNA with IRE1α peptide

1. IRE1α protein, active (SignalChem, catalog number: E31-11G) (store at -80 °C)

2. IRE1α inhibitor 4μ8C (MedChemExpress, catalog number: HY-19707) (store at -20 °C)

3. RNase A 100 mg/mL (QIAGEN, catalog number: 1007885) (store at -20 °C; shelf life: 12 months)

4. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014) (store at room temperature)

5. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266) (Store at room temperature)

6. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) (Store at -20 °C)

7. Glycerol (Sigma-Aldrich, catalog number: G5516) (Store at room temperature)

8. Adenosine 5’-triphosphate (ATP) (Sigma-Aldrich, catalog number: A2383) (Store at -20 °C)

C. cDNA synthesis after RNA cleavage

1. PrimeScript^TM RT Reagent kit (Takara, catalog number: RR037A) (store at -20 °C)

D. Checking mRNA expression of RIDD target mRNA

1. Solg^TM 2× Taq PCR Smart mix 1 (SolGent Co. Ltd., catalog number: STD01-M50h) (store at -20 °C)

2. 100 bp DNA ladder (Bioneer Corporation, catalog number: D-1035) (store at -20 °C)

3. Ultrapure^TM agarose (Invitrogen^TM, catalog number: 16500-100) (store at room temperature)

E. qRT-PCR

1. Power SYBR^TM Green PCR Master Mix (Applied Biosystems^TM, catalog number: 4367659) (store at -20 °C)

2. 1 M HEPES buffer pH 7.4 (Bio-solution Co., Ltd., catalog number: BH048) (store at room temperature)

3. 50× TAE buffer (Bio-Rad Laboratories, Inc., catalog number: 1610743) (store at room temperature)

4. UltraPure^TM ethidium bromide, 10 mg/mL (Invitrogen^TM, catalog number: 15585011) (store at room temperature)

Solutions

1. 1 M NaCl (see Recipes)

2. 100 mM ATP (see Recipes)

3. 1 M MgCl₂ (see Recipes)

4. 1 M DTT (see Recipes)

5. 5× Reaction buffer (see Recipes)

6. 0.5× TAE buffer (see Recipes)

Recipes

1. 1 M NaCl

Reagent	Final concentration	Quantity or Volume
NaCl	1 M	58.44 mg/1 mL
Ultrapure^TM DNase/RNase-free distilled water	-	1 mL
Storage condition: Room temperature; shelf life: 1 month

2. 100 mM ATP

Reagent	Final concentration	Quantity or Volume
ATP	100 mM	55.114 mg/1 mL
Ultrapure^TM DNase/RNase-free distilled water	-	1 mL
Storage condition: -20 °C, avoid repeated freeze-thaw cycles; shelf life: 1 month

3. 1 M MgCl₂

Reagent	Final concentration	Quantity or Volume
MgCl₂	1 M	95.21 mg/1 mL
Ultrapure^TM DNase/RNase-free distilled water	-	1 mL
Storage condition: Room temperature; shelf life: 1 month

4. 1 M DTT

Reagent	Final concentration	Quantity or Volume
DTT	1 M	15.425 mg/0.1 mL
Ultrapure^TM DNase/RNase-free distilled water	-	0.1 mL
Storage condition: -20 °C, avoid repeated freeze-thaw cycles; shelf life: 1 month

5. 5× Reaction buffer

Reagent	Final concentration	Quantity or Volume
1 M HEPES pH 7.4	100 mM	10 μL
1 M NaCl	350 mM	35 μL
100 mM ATP	10 mM	10 μL
1 M MgCl₂	10 mM	1 μL
1 M DTT	25 mM	2.5 μL
Glycerol	25 % v/v	25 μL
Ultrapure^TM DNase/RNase-free distilled water	-	16.5 μL
Total volume		100 μL
*5× reaction buffer is prepared freshly for every experiment.

6. 0.5× TAE buffer

Reagent	Final concentration	Quantity or Volume
50× TAE buffer	0.5×	10 mL
Distilled water	-	990 mL
Total volume		1 L
Storage condition: Room temperature; shelf life: 1 month.

Laboratory supplies

1. RNase-free 1.5 mL microcentrifuge tubes (Corning Life Sciences Co., Ltd, catalog number: MCT-150-C)

2. Pipette tips 1 mL (Corning Life Sciences Co., Ltd, catalog number: T-1000-B)

3. Pipette tips 200 μL (Corning Life Sciences Co., Ltd, catalog number: T-200-C)

4. Pipette tips 10 μL (Corning Life Sciences Co., Ltd, catalog number: T-300)

5. PCR tubes (Hundaimicro, catalog number: 203017)

6. MicroAmp^TM optical 384-well reaction plate with barcode (Applied Biosystem^TM, catalog number: 4309849)

7. MicroAmp^TM optical adhesive film (Applied Biosystem^TM, catalog number: 4311971)

Equipment

1. Veriti 96-well thermal cycler (Applied Biosystem, model: 9902)

2. -80 °C freezer (Nihon Freezer Co., LTD., model: CLN-71UWM)

3. Fresco^TM 17 microcentrifuge (Thermo Scientific^TM, catalog number: 75002402)

4. Mupid-2plus electrophoresis system (Takara, model: AD110)

5. DeNovix spectrophotometer (DeNovix Inc., model: DS-11+)

6. UltraSlim UV transilluminator (Maestrogen Inc., model: UUV-01)

7. Agilent 2100 bioanalyzer (Agilent Technologies, model: G2939B)

8. QuantStudio^TM 6 Flex real-time PCR systems, 384-well, laptop (Applied Biosystem^TM, model: 4485691)

Software and datasets

1. ImageJ image processing software (NIH, USA, 1.53e)

2. GraphPad Prism 10 (GraphPad Software, LLC, Boston, USA, 10.2.3)

Procedure

English

中文翻译

文章信息

稿件历史记录

提交日期: Apr 21, 2025

接收日期: Jul 15, 2025

在线发布日期: Jul 24, 2025

出版日期: Aug 20, 2025

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如何引用

Rashid, M. M. U., Rah, S., Ko, S. and Chae, H. (2025). An Ex Vivo Protocol to Assess IRE1α-Dependent RNA Cleavage Using Total RNA Isolated from Mouse Tissues. Bio-protocol 15(16): e5414. DOI: 10.21769/BioProtoc.5414.