Decay Kinetics of Autogenously Regulated CGS1 mRNA That Codes for Cystathionine γ-Synthase in Arabidopsis thaliana

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Abstract
Cystathionine γ-synthase (CGS) catalyses the first committed step in methionine (Met) biosynthesis in higher plants. Stability of CGS1 mRNA encoding CGS in Arabidopsis thaliana is regulated by negative feedback in response to Met application and the amino acid sequence of CGS itself acts in cis in this regulation. It is proposed that the regulation occurs during translation when the nascent polypeptide of CGS and its mRNA are in close proximity. This model predicts that inhibition of translation abolishes the regulation. To test this, we analysed the effect of translation inhibitor cycloheximide on the CGS1 mRNA decay. The half-life of CGS1 mRNA after the addition of transcription inhibitor actinomycin D in the absence and presence of 1 mM Met was 154±11 min and 81±5 min, respectively. Simultaneous addition of actinomycin D and cycloheximide stabilized CGS1 mRNA both in the presence and absence of Met, as essentially no decrease of CGS1 mRNA was observed. Moreover, cycloheximide treatment inhibited production of the truncated CGS1 RNA species, a possible degradation intermediate. These results indicated that inhibition of translation abolishes the CGS1 mRNA-specific decay process. Kinetic analyses indicated that about half the CGS1 mRNA is destined to CGS1 mRNA-specific decay when 1 mM Met was applied.