ABSTRACT
Overexpression of S-adenosylmethionine decarboxylase (SAMDC) mRNA in 1- and 2-cell stage Xenopus embryos induces cell autonomous dissociation at the late blastula stage and developmental arrest at the early gastrula stage. The induction of cell dissociation took place "punctually" at the late blastula stage in the SAMDC-overexpressing cells, irrespective of the stage of the microinjection of SAMDC mRNA. When we examined the cells undergoing the dissociation, we found that they were TUNEL-positive and contained fragmented nuclei with condensed chromatin and fragmented DNA. Furthermore, by injecting Xenopus Bcl-2 mRNA together with SAMDC mRNA, we showed that SAMDC-overexpressing embryos are rescued completely by Bcl-2 and becometadpoles. These results indicatethat cell dissociation induced by SAMDC overexpression is due to apoptotic cell death. Since the level of S-adenosylmethionine (SAM) is greatly reduced in SAMDC-overexpressing embryos and this induces inhibition of protein synthesis accompanied by the inhibition of DNA and RNA syntheses, we conclude that deficiency in SAM induced by SAMDC overexpression activates the maternal program of apoptosis in Xenopus embryos at the late blastula stage, but not before. We propose that this mechanism serves as a surveillance mechanism to check and eliminate cells physiologically damaged during the cleavage stage.
Subject(s)
Adenosylmethionine Decarboxylase/metabolism , Embryo, Nonmammalian/metabolism , Animals , Blastocyst/metabolism , DNA/metabolism , DNA, Complementary/metabolism , Electrophoresis, Agar Gel , Embryo, Nonmammalian/ultrastructure , In Situ Nick-End Labeling , Microinjections , Microscopy, Electron , Plasmids/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/metabolism , S-Adenosylmethionine/metabolism , Time Factors , XenopusABSTRACT
When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.
Subject(s)
Adenosylmethionine Decarboxylase/metabolism , Apoptosis , Blastocyst/physiology , Xenopus/embryology , Adenosylmethionine Decarboxylase/genetics , Animals , Blastocyst/ultrastructure , Microinjections , Models, Biological , RNA, Messenger/genetics , RNA, Messenger/metabolism , Time FactorsABSTRACT
The mRNA cap structure, which is synthesized by a series of reactions catalyzed by capping enzyme, mRNA (guanine-7-)-methyltransferase, and mRNA (ribose-2'-O-)-methyltransferase, has crucial roles for RNA processing and translation. Methylation of the cap structure is also implicated in polyadenylation-mediated translational activation during Xenopus oocyte maturation. Here we isolated two Xenopus laevis cDNAs, xCAP1a and xCAP1b, for mRNA capping enzyme and one cDNA for mRNA (guanine-7-)-methyltransferase, xCMT1, which encode 598, 511, and 402 amino acids, respectively. The deduced amino acid sequence of xCAP1a was highly homologous to that of human capping enzyme hCAP1a, having all the characteristic regions including N-terminal RNA 5'-triphosphatase as well as C-terminal mRNA guanylyltransferase domains which are conserved among animal mRNA guanylyltransferases, whereas in xCAP1b the most C-terminal motif was missing. The amino acid sequence of xCMT1 was also similar to human (guanine-7-)-methyltransferase, hCMT1a, with all the conserved motifs among cellular (guanine-7-)-methyltransferases, except for its N-terminal portion. The recombinant xCAP1a and xCMT1 exhibited cap formation and mRNA (guanine-7-)-methyltransferase activities, respectively. RT-PCR analysis showed that mRNA for xCAP1a and xCMT1 exist abundantly in fertilized eggs as maternal mRNAs, but xCMT1 mRNA gradually decreased in its amount in later stages of early development.
