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Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis.

ABSTRACT: Plant and metazoan microRNAs (miRNAs) guide ARGONAUTE (AGO) protein complexes to regulate expression of complementary RNAs via base pairing. In the plant Arabidopsis thaliana, the main miRNA effector is AGO1, but few other factors required for miRNA activity are known. Here, we isolate the genes defined by the previously described miRNA action deficient (mad) mutants, mad3 and mad4. Both genes encode enzymes involved in isoprenoid biosynthesis. MAD3 encodes 3-hydroxy-3-methylglutaryl CoA reductase (HMG1), which functions in the initial C(5) building block biogenesis that precedes isoprenoid metabolism. HMG1 is a key regulatory enzyme that controls the amounts of isoprenoid end products. MAD4 encodes sterol C-8 isomerase (HYDRA1) that acts downstream in dedicated sterol biosynthesis. Using yeast complementation assays and in planta application of lovastatin, a competitive inhibitor of HMG1, we show that defects in HMG1 catalytic activity are sufficient to inhibit miRNA activity. Many isoprenoid derivatives are indispensable structural and signaling components, and especially sterols are essential membrane constituents. Accordingly, we provide evidence that AGO1 is a peripheral membrane protein. Moreover, specific hypomorphic mutant alleles of AGO1 display compromised membrane association and AGO1-membrane interaction is reduced upon knockdown of HMG1/MAD3. These results suggest a possible basis for the requirement of isoprenoid biosynthesis for the activity of plant miRNAs, and unravel mechanistic features shared with their metazoan counterparts.

SUBMITTER: Brodersen P

PROVIDER: S-EPMC3277166 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis.

Brodersen Peter P Sakvarelidze-Achard Lali L Schaller Hubert H Khafif Mehdi M Schott Grégory G Bendahmane Abdelhafid A Voinnet Olivier O

Proceedings of the National Academy of Sciences of the United States of America 20120112 5

Plant and metazoan microRNAs (miRNAs) guide ARGONAUTE (AGO) protein complexes to regulate expression of complementary RNAs via base pairing. In the plant Arabidopsis thaliana, the main miRNA effector is AGO1, but few other factors required for miRNA activity are known. Here, we isolate the genes defined by the previously described miRNA action deficient (mad) mutants, mad3 and mad4. Both genes encode enzymes involved in isoprenoid biosynthesis. MAD3 encodes 3-hydroxy-3-methylglutaryl CoA reducta ...[more]

PMID: 22247288

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Project description:In plants, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of isoprenoids, is compartmentalized: the mevalonate (MVA) pathway, which is localized to the cytosol, is responsible for the synthesis of sterols, certain sesquiterpenes, and the side chain of ubiquinone; in contrast, the recently discovered MVA-independent pathway, which operates in plastids, is involved in providing the precursors for monoterpenes, certain sesquiterpenes, diterpenes, carotenoids, and the side chains of chlorophylls and plastoquinone. Specific inhibitors of the MVA pathway (lovastatin) and the MVA-independent pathway (fosmidomycin) were used to perturb biosynthetic flux in Arabidopsis thaliana seedlings. The interaction between both pathways was studied at the transcriptional level by using GeneChip (Affymetrix) microarrays and at the metabolite level by assaying chlorophylls, carotenoids, and sterols. Treatment of seedlings with lovastatin resulted in a transient decrease in sterol levels and a transient increase in carotenoid as well as chlorophyll levels. After the initial drop, sterol amounts in lovastatin-treated seedlings recovered to levels above controls. As a response to fosmidomycin treatment, a transient increase in sterol levels was observed, whereas chlorophyll and carotenoid amounts decreased dramatically when compared with controls. At 96 h after fosmidomycin addition, the levels of all metabolites assayed (sterols, chlorophylls, and carotenoids) were substantially lower than in controls. Interestingly, these inhibitor-mediated changes were not reflected in altered gene expression levels of the genes involved in sterol, chlorophyll, and carotenoid metabolism. The lack of correlation between gene expression patterns and the accumulation of isoprenoid metabolites indicates that posttranscriptional processes may play an important role in regulating flux through isoprenoid metabolic pathways.

Dataset Information

Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis.

Publications

Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis.

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