Chiba Yukako, Sakurai Ryoko, Yoshino Michiko, Ominato Kimihiro, Ishikawa Mari, Onouchi Hitoshi, Naito Satoshi
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10225-30. doi: 10.1073/pnas.1831512100. Epub 2003 Aug 21.
Cystathionine gamma-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, S-adenosyl-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. S-adenosyl-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.
胱硫醚γ-合酶是高等植物甲硫氨酸生物合成的首个关键酶,在拟南芥中由CGS1基因编码。我们之前已经表明,CGS1 mRNA的稳定性会因甲硫氨酸的施用而受到负调控[千叶洋、石川真、木岛芳、泰森·R·H、金杰、山本明、南原英、勒斯泰克、R·M·沃尔斯格罗夫和内藤史(1999年)《科学》286卷,第1371 - 1374页]。为了确定甲硫氨酸本身是否是CGS1外显子1介导的转录后调控的效应物,我们进行了转染实验。结果表明,作为这种调控效应物的是S-腺苷-L-甲硫氨酸(AdoMet)或其代谢产物之一,而非甲硫氨酸。为了进一步确定实际的效应物,我们利用了小麦胚体外翻译系统。通过使用携带CGS1外显子1-报告基因融合体的RNA,测试了AdoMet的各种代谢产物和类似物的作用。这些测试确定AdoMet是这种调控的效应物。AdoMet的类似物S-腺苷-L-乙硫氨酸也具有效应物活性。缺乏这种调控的拟南芥mto1突变体在体外对AdoMet的反应大幅降低,而一个渗漏等位基因的反应降低程度较小。在AdoMet存在的情况下体外翻译的RNA包含一种5'端截短的RNA种类,类似于我们之前认为的CGS1 mRNA在体内的降解中间体。总之,结果表明CGS1外显子1介导的转录后调控的基本反应在小麦胚体外翻译系统中发生,并且AdoMet作为效应物起作用。