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ETHYLENE-INSENSITIVE5 encodes a 5' to 3' exoribonuclease required for posttranscriptional regulation

ABSTRACT: Ethylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F box proteins, EBF1 and EBF2 (1-3). Here, we report the identification of ETHYLENE INSENSITIVE5 as the 5’?3’ exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response. Keywords: total RNA profiling, proteolysis, plant hormoine, ethylene

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE5174 | GEO | 2006/08/08

SECONDARY ACCESSION(S): PRJNA96587

REPOSITORIES: GEO

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Project description:Ethylene is a gaseous plant growth regulator that controls a multitude of developmental and stress responses. Recently, the levels of Arabidopsis EIN3 protein, a key transcription factor mediating ethylene-regulated gene expression, have been demonstrated to increase in response to the presence of ethylene gas. Furthermore, in the absence ethylene, EIN3 is quickly degraded through a ubiquitin/proteasome pathway mediated by two F box proteins, EBF1 and EBF2 (1-3). Here, we report the identification of ETHYLENE INSENSITIVE5 as the 5â?3â exoribonuclease XRN4. Specifically, we demonstrate that EIN5 is a component of the ethylene signal transduction cascade acting downstream of CTR1 that is required for ethylene-mediated gene expression changes. Furthermore, we find that the ethylene insensitivity of ein5 mutant plants is a consequence of the over-accumulation of EBF1 and EBF2 mRNAs resulting in the under-accumulation of EIN3 even in the presence of ethylene gas. Together, our results suggest that the role of EIN5 in ethylene perception is to antagonize the negative feedback regulation on EIN3 by promoting EBF1 and EBF2 mRNA decay, which consequently allows the accumulation of EIN3 protein to trigger the ethylene response. Experiment Overall Design: Cold-treated seeds in MS plates were placed in chambers at 24oC in the dark with hydrocarbon-free airflow for three days, after which some of the chambers were connected to ethylene gas at 10 ppm while the others remained on air treatment. Four hours later the seedlings of each plate were quickly collected and frozen in liquid nitrogen. Total RNA was prepared from both air-treated and ethylene-treated etiolated seedlings of Arabidopsis Col-0, ein2-5 and ein5-1 using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Biotinylated target RNA was prepared from 16 ?g of total RNA using the procedure described by the manufacturer (Affymetrix, Santa Clara, CA). Briefly, a primer encoding a T7 RNA polymerase promoter fused to (dT)24 (Genset Oligos, La Jolla, CA) was used to prime double-stranded cDNA synthesis using the SuperScript Choice System (Life Technologies). The resulting cDNA was transcribed in vitro using the BioArray High-Yield RNA Tran-script Labeling Kit (Enzo Biochem, New York, NY) in the presence of biotinylated UTP and CTP to produce biotinylated target complementary RNA (cRNA). The labeled target cRNA was purified, fragmented, and hybridized to Arabidopsis microoarrays (Affymetrix whole genome tiling, and ATH1 gene expression arrays) according to protocols provided by the manufacturer in a hybridization oven model 640 (Affymetrix, Santa Clara, CA). The arrays were washed and stained with streptavidin-phycoerythrin using a GeneChip Fluidics Station model 400 and then scanned with a Gene Array Scanner (Hewlett-Packard, Palo Alto, CA). Scanned images were processed and quantified using GeneChip Suite 3.2. Genespring software (Silicon Genetics, Redwood City, CA) was used to manage and filter the array data. Each measurement was divided by the 50.0th percentile of all measurements in that sample. The percentile was calculated with all normalized measurements above 10. For samples where the bottom tenth percentile was less than the negative of the 50.0th percentile, it was used as a background, and subtracted from all the other genes first. Experiments were carried out in duplicate and hybridized to two sets of expression chips. Expression values were analyzed using GeneSpringâ¢ software version 4.2 (Silicon Genetics, Redwood, California).

Project description:Whole genome small RNA sequencing (wild-type Col-0, abh1-1, ein5-6, ein5-6 abh1-1) and 5'-RACE (GMUCT) (wild-type Col-0 and ein5-6) sequencing of parenthetically indicated genotypes of Arabidopsis using the Illumina Genetic Analyzer. Whole-genome oligonucleotide tiling microarrays were used for gene expression studies of the entire transcriptome for wildtype Col-0, abh1-1, ein5-6, and ein5-6 abh1-1 plants. Micro (mi)RNAs and small-interfering (si)RNAs are abundant endogenous small (sm)RNAs that control transcript expression by post-transcriptional gene silencing. Here, we show that concomitant loss of XRN4/EIN5, a 5’-3’ exoribonuclease, and CBP80/ABH1, the largest subunit of the mRNA cap binding complex, results in Arabidopsis plants manifesting myriad developmental defects. Through the analysis of ein5 abh1 double mutant plants, we find that CBP80/ABH1 regulates the levels of mature miRNAs, which suggests this protein is a novel component of the miRNA-mediated RNA silencing pathway. Additionally, we show that a novel class of smRNAs are processed from both sense and anti-sense strands of ~130 endogenous transcripts that apparently are converted to double-stranded RNA and subsequently processed into smRNAs, and accumulate in the absence of XRN4/EIN5. Moreover, we demonstrate that accumulation of these smRNAs is often synergistically increased in ein5 abh1 double mutant plants, which suggests that these proteins act coordinately to regulate the substrates from which they are processed. Finally, we find that the parent transcripts of these novel smRNAs accumulate in an uncapped form upon loss of XRN4/EIN5. These results suggest that uncapped endogenous transcripts can shuttle into an RNA silencing pathway where they become smRNA biogenesis substrates. Overall, our results reveal unexpected connections between RNA metabolism and silencing. Keywords: transcriptome analysis using tiling array; whole genome small RNA sequencing; 5'-RACE sequencing

Project description:One of the major players controlling RNA decay is the cytoplasmic 5’ to 3’ exoribonuclease, which is conserved among eukaryotic organisms. In Arabidopsis, the 5’ to 3’ exoribonuclease XRN4 is involved in disease resistance, the response to ethylene, RNAi and miRNA-mediated RNA decay. Curiously, XRN4 appears to display selectivity among its substrates because certain 3’ cleavage products formed by miRNA-mediated decay, such as from ARF10 mRNA, accumulate in the xrn4 mutant, whereas others, such as from AGO1, do not. To examine the nature of this selectivity, transcripts that differentially accumulate in xrn4 were identified by combining PARE and Affymetrix arrays. Certain functional categories, such as stamen-associated proteins and hydrolases, were over-represented among transcripts decreased in xrn4, whereas transcripts encoding nuclear-encoded chloroplast-targeted proteins and nucleic acid-binding proteins were over-represented in transcripts increased in xrn4. To ascertain if RNA sequence influences the apparent XRN4 selectivity, a series of chimeric constructs were generated where the miRNA-complementary sites and different portions of the surrounding sequences from AGO1 and ARF10 were interchanged. Analysis of the resulting transgenic plants revealed that the presence of a 150 nucleotide sequence downstream of the ARF10 miRNA-complementary site conferred strong accumulation of the 3’ cleavage products in xrn4. In addition, sequence analysis of differentially accumulating transcripts led to the identification of 27 hexamer motifs that were over-represented in transcripts or miRNA-cleavage products accumulating in xrn4. Taken together, the data indicate that specific mRNA sequences, like those in ARF10, and mRNAs from select functional categories are attractive targets for XRN4-mediated decay. Mixed-stage inflorescences from Col-0 and xrn4-5 plants were harvested. Total RNA was extracted and hybridized to the ATH1 array to determine genes differentially expressed in xrn4-5 compared to Col-0. 2 replicates each.

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ETHYLENE-INSENSITIVE5 encodes a 5' to 3' exoribonuclease required for posttranscriptional regulation

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