Identification of a Short Highly Conserved Amino Acid Sequence as the Functional Region Required for Posttranscriptional Autoregulation of the Cystathionine γ-Synthase Gene in Arabidopsis

Abstract

Cystathionine ␥-synthase (CGS) catalyzes the first committed step of Met biosynthesis in plants. We have previously shown that expression of the gene for CGS is feedback-regulated at the level of mRNA stability, and that the amino acid sequence encoded by the first exon of the CGS gene itself is responsible for the regulation (Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J., Yamamoto, A., Nambara, E., Leustek, T., Wallsgrove, R. M., and Naito, S. (1999) Science 286, 1371-1374). To identify the functional region within CGS exon 1, deletion analysis was performed. The results showed that the 41-amino acid region of exon 1 highly conserved among plants is necessary and sufficient for the regulation. Analyses of in vivo and in vitro generated mutations that abolish the regulation identified the functionally important amino acids as 11-13 residues within this conserved region. The importance of these residues was confirmed by deletion analysis within the conserved region. These studies identified the functional region of CGS exon 1 required for the posttranscriptional autoregulation of the CGS gene as (A)RRNCSNIGVAQ(I), with uncertainty of the first and last residues. This sequence is almost perfectly conserved among CGS sequences of higher plants but cannot be found elsewhere in the public databases.

Dynamics of methionine biosynthesis

“…All subsequent mto1 alleles were also found to be located within this region. Alaninesubstitution experiments identified a short stretch of amino acid sequence, Arg-Arg-Asn-Cys-Ser-Asn-IleGly-Val-Ala-Gln, that covers the mto1 mutation sites that is important for the regulation, which is now referred to as the MTO1 region (Ominato et al 2002) (Figure 3). As a result of the mutation within the CGS exon 1 coding sequence, CGS mRNA levels were two-to four-fold higher in mto1 than in wild-type plants, which is reflected by elevated levels of CGS protein and enzyme activity (Chiba et al 1999;Ominato et al 2002).…”

“…Alaninesubstitution experiments identified a short stretch of amino acid sequence, Arg-Arg-Asn-Cys-Ser-Asn-IleGly-Val-Ala-Gln, that covers the mto1 mutation sites that is important for the regulation, which is now referred to as the MTO1 region (Ominato et al 2002) (Figure 3). As a result of the mutation within the CGS exon 1 coding sequence, CGS mRNA levels were two-to four-fold higher in mto1 than in wild-type plants, which is reflected by elevated levels of CGS protein and enzyme activity (Chiba et al 1999;Ominato et al 2002). Analysis of the regulation defect in mto1 revealed that wild-type CGS is negatively regulated in response to exogenous application of methionine, and that posttranscriptional regulation at the level of mRNA stability plays a major role (Chiba et al 1999).…”

“…Identical amino acid residues are reversed and similar ones are shaded. The MTO1 region marked below the alignment was identified by alaninesubstitution experiments (Ominato et al 2002). Filled arrowhead indicates the position of translation arrest induced by SAM.…”

A halt in poly(A) shortening during <i>S</i>-adenosyl-L-methionine-induced translation arrest in <i>CGS1</i> mRNA of <i>Arabidopsis thaliana</i>

“…A. thaliana mto1 mutants that bear single amino acid sequence alterations within the MTO1 region abolish this feedback regulation, resulting in an over-accumulation of soluble Met (Ominato et al, 2002; for review, see Goto et al, 2005).…”

“…A short amino acid sequence, termed the MTO1 region, 77-RRNCSNIGVAQ-87, encoded in the N-terminal region of CGS1, acts as the cis-element for this regulation (Ominato et al, 2002;Chiba et al, 2003). A. thaliana mto1 mutants that bear single amino acid sequence alterations within the MTO1 region abolish this feedback regulation, resulting in an over-accumulation of soluble Met (Ominato et al, 2002; for review, see Goto et al, 2005).…”