Expression of the Arabidopsis CGS1 gene that codes for cystathionine ␥-synthase is feedback regulated at the step of mRNA stability in response to S-adenosyl-L-methionine (AdoMet). A short stretch of amino acid sequence, called the MTO1 region, encoded by the first exon of CGS1 itself is involved in this regulation. Here, we demonstrate, using a cell-free system, that AdoMet induces temporal translation elongation arrest at the Ser-94 codon located immediately downstream of the MTO1 region, by analyzing a translation intermediate and performing primer extension inhibition (toeprint) analysis. This translation arrest precedes the formation of a degradation intermediate of CGS1 mRNA, which has its 5 end points near the 5 edge of the stalled ribosome. The position of ribosome stalling also suggests that the MTO1 region in nascent peptide resides in the ribosomal exit tunnel when translation elongation is temporarily arrested. In addition to the MTO1 region amino acid sequence, downstream Trp-93 is also important for the AdoMet-induced translation arrest. This is the first example of nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in eukaryotes. Furthermore, our data suggest that the ribosome stalls at the step of translocation rather than at the step of peptidyl transfer.[Keywords: S-adenosyl-L-methionine; translation arrest; mRNA stability; MTO1 region; cystathionine ␥-synthase; feedback regulation] Supplemental material is available at http://www.genesdev.org. Cystathionine ␥-synthase (CGS; EC 2.5.1.48) catalyzes the first committed step of methionine biosynthesis in higher plants (Matthews 1999) and is encoded by the CGS1 gene in Arabidopsis thaliana (gene ID At3g01120; Kim and Leustek 1996). Expression of the CGS1 gene is regulated by negative feedback at the step of mRNA stability in response to methionine application in Arabidopsis. When wild-type calli were treated with methionine, the amount of full-length CGS1 mRNA was decreased, and a short CGS1 RNA species formed that is truncated at its 5Ј region. This 5Ј-truncated RNA species is likely an intermediate of CGS1 mRNA degradation. The mto1 mutants of Arabidopsis, which carry single amino acid sequence alterations within the first exon of CGS1, are deficient in this regulation and overaccumulate soluble methionine (Chiba et al. 1999). A short stretch of amino acid sequence, termed the MTO1 region, which is encoded within CGS1 exon 1 and covers the mto1 mutation sites, is involved in this process (Ominato et al. 2002). Since CGS1 exon 1 acts in cis, we proposed that this regulation occurs during translation, when the nascent polypeptide and its mRNA are in close proximity (Chiba et al. 1999;Suzuki et al. 2001). In support of this idea, the regulation is abolished by application of a translation inhibitor (Lambein et al. 2003).CGS1 exon 1-mediated post-transcriptional regulation was reproduced in an in vitro translation system using wheat germ extract, and S-adenosyl-L-methionine (AdoMet), a direct metabolite of methionine...

The Fas antigen (Fas) is a cell‐surface receptor protein that mediates apoptosis‐inducing signals and plays an important role in the immune system. Significant amounts of Fas mRNA can be detected not only in lymphoid organs but also in the liver, heart, and ovary. In the ovary, apoptosis is thought to cause follicular atresia and luteolysis. We have investigated the involvement of Fas in these events. Here we report that Fas protein is expressed on granulosa and luteal cells but not on oocytes in the ovary. An injection of anti‐Fas monoclonal antibody with apoptosis‐inducing activity into adult mice enhanced follicular atresia and luteolysis. After the injection, the corpora lutea disappeared and the number of follicles containing pyknotic granulosa cells increased. There were also fewer ovulated ova and lower levels of luteal cell‐produced progesterone. Furthermore, as the result of a non‐functional Fas/Fas ligand system, mature ovaries from the mouse mutant lpr (lymphoproliferation) were histologically abnormal in terms of follicular development, in that the number of secondary follicles significantly increased. These results suggested that Fas plays an important role in follicular atresia and luteolysis in the ovarian physiology of adult mice. Mol. Reprod. Dev. 47:11–18, 1997. © 1997 Wiley‐Liss, Inc.

ABSTRACT

SummaryRecent studies have revealed that 10-20% of CD4+8 -or CD4-8-thymocyte populations contain NKI.1 + T cell receptor (TCR)-cff~ + cells. This subpopulation shows characteristics that are different from NKI.1-CD4 § or NKI.1-CD8 + T calls and seems to have developed in a manner different from NKI.1-T cells. Although extensive studies have been performed on the NKI.1 + TCR-od~8 + thymocytes, the physiological role of the NKI.1 + TCR-c~/~/+ thymocytes has been totally unclear. In the present study, we found that fleshly isolated NKI.1 + TCR-odB + thymocytes, but neither whole thymocytes nor lymph node T cells, directly killed CD4 +8 § thymocytes from normal syngeneic or allogeneic mice by using a long-term cytotoxic assay in which flow cytometry was used to detect the cytotoxidty. However, only weak cytotoxicity was detected against thymocytes from lpr mice on which the Fas antigen that transduces signals for apoptosis into the cells is not expressed. Furthermore, the NKI.1 + TCR-ot/B + thymocytes exhibited high cytotoxicity against T lymphoma targets transfected withj~s genes as compared with the parental T lymphoma targets or target cells transfected with mutatedJas genes, which lack the function of transducing signals. On the other hand, NKI.1 § effector thymocytes from gld mice that carry a point mutation in Fas ligand did not kill thymocyte targets from normal mice. The present findings, thus, consistently suggest that the NKI.1 + TCR-ot/~8 + thymocytes kill a subpopulation among CD4+8 + thymocytes via Fas antigen and in this way regulate generation of T lineage cells in the thymus.

No abstract