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Published on: December 13, 2014
Characterization of IRE1 ribonuclease-mediated mRNA decay in plants using transient expression analyses in rice
Shimpei Hayashi1, Yuhya Wakasa1, Kenjirou Ozawa1
1Genetically Modified Organism Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan.
Abstract:
In some eukaryotes, endoplasmic reticulum (ER) stress induces regulated inositol-requiring enzyme 1 (IRE1)-dependent decay (RIDD) of mRNAs. Recently, the expression levels of the mRNAs encoding some secretory proteins were reported to be downregulated by RIDD in the vegetative tissues of plants. However, the characteristics of plant RIDD have been insufficiently investigated due to difficulty of in planta analyses. Here, the RIDD susceptibilities of various mRNAs that are difficult to analyze in planta were examined using transient expression analyses of rice protoplasts. In this system, the mRNAs encoding three rice seed storage proteins (SSPs) - namely α-globulin, 16-kDa prolamin and 10-kDa prolamin - were downregulated in response to ER stress. The rapid ER stress-induced degradation of these mRNAs was repressed in cells in which the ribonuclease activity of IRE1 was specifically abolished by genome editing, suggesting that the mRNAs encoding certain SSPs are strong targets of RIDD. Furthermore, we investigated whether these RIDD targets are substrates of the IRE1 ribonuclease using a recombinant IRE1 protein, and identified candidate IRE1-mediated cleavage sites. Overall, the results demonstrate the existence of a post-transcriptional mechanism of regulation of SSPs, and illustrate the basic and multifaceted characteristics of RIDD in higher plants.
Insights
Endoplasmic reticulum (ER) stress triggers regulated inositol-requiring enzyme 1 (IRE1)-dependent decay (RIDD) in plants. This study shows RIDD degrades seed storage protein mRNAs in rice, revealing a key post-transcriptional regulation mechanism.
Area of Science:
- Molecular Biology
- Plant Science
- Biochemistry
Background:
- Endoplasmic reticulum (ER) stress is a cellular response conserved across eukaryotes.
- Regulated inositol-requiring enzyme 1 (IRE1)-dependent decay (RIDD) is an ER stress-induced mRNA degradation pathway.
- Plant RIDD has been poorly characterized due to challenges in in planta analysis.
Purpose of the Study:
- To investigate the characteristics and targets of RIDD in plants.
- To examine the susceptibility of various mRNAs to RIDD using a transient expression system.
- To elucidate the role of IRE1 ribonuclease activity in plant RIDD.
Main Methods:
- Transient expression analysis in rice protoplasts.
- Genome editing to abolish IRE1 ribonuclease activity.
- Recombinant IRE1 protein assays to identify cleavage sites.
Main Results:
- ER stress downregulated mRNAs encoding three rice seed storage proteins (SSPs) in protoplasts.
- Degradation of SSP mRNAs was dependent on IRE1 ribonuclease activity.
- Candidate IRE1-mediated cleavage sites were identified in SSP mRNAs.
Conclusions:
- Plant RIDD targets specific mRNAs, including those for seed storage proteins.
- IRE1 ribonuclease activity is crucial for RIDD-mediated mRNA degradation in plants.
- This study reveals a post-transcriptional regulatory mechanism for SSPs in plants and characterizes plant RIDD.
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