S-adenosyl- l -methionine is an effector in the posttranscriptional autoregulation of the cystathionine γ-synthase gene in Arabidopsis

Abstract

Cystathionine ␥-synthase, the first committed enzyme of methionine biosynthesis in higher plants, is encoded by the CGS1 gene in Arabidopsis thaliana. We have shown previously that the stability of the CGS1 mRNA is negatively regulated in response to methionine application [Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J., Yamamoto, A., Nambara, E., Leustek, T., Wallsgrove, R. M. & Naito, S. (1999) Science 286, 1371-1374]. To determine whether methionine itself is the effector of the CGS1 exon 1-mediated posttranscriptional regulation, we carried out transfection experiments. The results suggested that, rather than methionine, Sadenosyl-L-methionine (AdoMet), or one of its metabolites, acts as the effector of this regulation. To further identify the actual effector, we exploited the wheat germ in vitro translation system. The effects of various metabolites and analogs of AdoMet were tested by using RNA carrying a CGS1 exon 1-reporter fusion. These tests identified AdoMet as the effector of this regulation. Sadenosyl-L-ethionine, an analog of AdoMet, also had effector activity. A. thaliana mto1 mutants, which are deficient in this regulation, showed a much reduced response to AdoMet in vitro, with a leaky allele showing a less reduced response. RNA translated in vitro in the presence of AdoMet contained a 5-truncated RNA species, similar to the one that we previously suggested was an in vivo degradation intermediate of CGS1 mRNA. Together, the results show that the basic reactions of CGS1 exon 1-mediated posttranscriptional regulation occur in the wheat germ in vitro translation system, and that AdoMet acts as the effector. C ystathionine ␥-synthase (CGS; EC 4.2.99.9) catalyzes the first committed step of methionine biosynthesis in higher plants (1) (Fig. 1), which is considered to be the key regulatory step in methionine biosynthesis (2-5). Unlike many of the key-step enzymes in metabolic pathways, CGS is not an allosteric enzyme (2). CGS is encoded in Arabidopsis thaliana by the CGS1 gene (gene ID At3g01120, GenBank accession no. AB010888) (6, 7). We have previously shown (8) that CGS1 expression involves feedback regulation at the level of mRNA stability in response to methionine application in vivo. A. thaliana mto1 mutants are deficient in this feedback regulation, and overaccumulate CGS1 mRNA, CGS protein, and soluble methionine. Seven independently isolated mto1 mutants were found to carry single-base changes within the first exon of CGS1, giving rise to amino acid sequence changes (8, 9).Transient and transgenic expression experiments using CGS1 exon 1-reporter fusions (8, 10) showed that the exon 1 coding sequence of CGS1 is necessary and sufficient for its posttranscriptional regulation, in response to exogenous application of methionine. In vitro mutagenesis of CGS1 exon 1 revealed that it is its amino acid sequence that has a role in this regulation. We have identified a stretch of 11-13 amino acid residues, termed the MTO1 region, located Ϸ80 residues from the N terminus of CGS, and coverin...

Dynamics of methionine biosynthesis

“…This fact provides a clear in vivo demonstration that TS This translation arrest triggers CGS mRNA degradation (Onouchi et al 2005). A 5Ј-truncated RNA species are formed as degradation intermediate (Chiba et al 1999;Chiba et al 2003) activity has a key role in regulating the carbon flow into the methionine biosynthetic pathway and, when TS activity is compromised, the regulation of CGS alone is not sufficient. This is in agreement with the finding that antisense repression of the TS gene in transgenic potato resulted in a reduction of TS activity to 6% of wild type and up to 230-fold increase in soluble methionine in leaf and tuber tissues, respectively (Zeh et al 2001).…”

“…Interestingly, although TS mRNA and protein levels were normal in mto2-1, accumulation of CGS mRNA and protein were negatively correlated with that of methionine and reduced to approximately 40% of that in wild type (Bartlem et al 2000) (Figure 5B). The reduced levels of CGS associated with methionine over-accumulation showed that the regulatory mechanism controlling CGS mRNA accumulation in response to increased SAM (Chiba et al 1999;Chiba et al 2003) was functioning normally. Methionine was able to over-accumulate 20-fold in mto2-1 when CGS levels were markedly reduced.…”

S -adenosylmethionine stabilizes cystathionine β-synthase and modulates redox capacity

“…In bacteria, AdoMet binds to RNA riboswitches and regulates genes involved in sulfur metabolism (13). In Arabidopsis thaliana, stability of the cystathionine ␥-synthase mRNA is negatively regulated by AdoMet (14), and in HepG2 cells, methionine withdrawal results in an AdoMet-dependent increase in MATII expression by stabilization of its mRNA (15). We excluded an effect of AdoMet on transcription (by Northern analysis and reporter assays using the Ϫ1b CBS promoter, which is most commonly used in liver) and translation (by in vitro transcription͞translation and polysome analysis) (data not shown).…”

Molecular Characterization of Barley Methionine γ-Lyase and Gene Expression by Abiotic Stress and Aspartate Family Amino Acids

“…Most methionine is converted to SAM by SAM synthase in plants [35]. SAM reduces the expression level of GCS in Arabidopsis [36]. SAM acts as a precursor for ethylene and polyamines biosynthesis, which supports tolerance against abiotic and biotic stresses [2,37,38].…”