RESUMEN
The formation of putrescine by ornithine decarboxylase (ODC) is a key regulatory step in polyamine biosynthesis in metazoa and fungi. Excess polyamines post-transcriptionally induce the synthesis of a unique non-competitive protein inhibitor of ODC, termed antizyme. Binding of antizyme to an ODC monomer subunit results in enzymatic inhibition, rapid ubiquitin-independent degradation of ODC by the 26S proteasome and recycling of antizyme. Plants possess an additional route for synthesizing putrescine via arginine decarboxylase (ADC). No homologue of ODC antizyme has been detected in plant genomes but several biochemical studies have reported plant ODC antizyme proteins of 9 and 16 kDa. Here we show that plant cells grown in liquid culture do not exhibit any substantial post-transcriptional, polyamine-responsive feedback regulation of ODC or ADC. However, using the yeast two hybrid system, a plant ODC-binding polypeptide was detected: the C-terminal 84-87 amino acids of cytosolic ribosomal protein (rp) S15. The Arabidopsis rpS15 polypeptide interacted specifically with plant ODC but not with human or Saccharomyces cerevisiae ODCs. Co-expression of either the full length or C-terminal rpS15 polypeptides with a plant ODC in yeast did not reduce ODC enzymatic activity. Only the full length mRNA encoding rpS15 was detected in Arabidopsis cells, suggesting that the C-terminal rpS15 polypeptide is encoded by a low abundance mRNA or the polypeptide is not physiologically relevant in plants. These results confirm the primacy of S-adenosylmethionine decarboxylase as the key regulatory enzyme in plant polyamine biosynthesis.
Asunto(s)
Citosol/metabolismo , Ornitina Descarboxilasa/metabolismo , Plantas/enzimología , Procesamiento Postranscripcional del ARN , Proteínas Ribosómicas/metabolismo , Secuencia de Aminoácidos , Línea Celular , Datos de Secuencia Molecular , Unión Proteica , ARN Mensajero/genética , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
Errors in chromosome segregation in mammalian oocytes lead to aneuploid eggs that are developmentally compromised. In mitotic cells, mitotic centromere associated kinesin (MCAK; KIF2C) prevents chromosome segregation errors by detaching incorrect microtubule-kinetochore interactions. Here, we examine whether MCAK is involved in spindle function in mouse oocyte meiosis I, and whether MCAK is necessary to prevent chromosome segregation errors. We find that MCAK is recruited to centromeres, kinetochores and chromosome arms in mid-meiosis I, and that MCAK depletion, or inhibition using a dominant-negative construct, causes chromosome misalignment. However, the majority of oocytes complete meiosis I and the resulting eggs retain the correct number of chromosomes. Moreover, MCAK-depleted oocytes can recover from mono-orientation of homologous kinetochores in mid-meiosis I to segregate chromosomes correctly. Thus, MCAK contributes to chromosome alignment in meiosis I, but is not necessary for preventing chromosome segregation errors. Although other correction mechanisms may function in mammalian meiosis I, we speculate that late establishment of kinetochore microtubules in oocytes reduces the likelihood of incorrect microtubule-kinetochore interactions, bypassing the requirement for error correction.
Asunto(s)
Aneuploidia , Proteínas de Ciclo Celular/metabolismo , Cinesinas/metabolismo , Meiosis , Oocitos/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Centrómero/metabolismo , Cromosomas de los Mamíferos/metabolismo , Cinesinas/genética , Cinetocoros/metabolismo , RatonesRESUMEN
A novel form of translational regulation is described for the key polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC). Plant AdoMetDC mRNA 5' leaders contain two highly conserved overlapping upstream open reading frames (uORFs): the 5' tiny and 3' small uORFs. We demonstrate that the small uORF-encoded peptide is responsible for constitutively repressing downstream translation of the AdoMetDC proenzyme ORF in the absence of increased polyamine levels. This first example of a sequence-dependent uORF to be described in plants is also functional in Saccharomyces cerevisiae. The tiny uORF is required for normal polyamine-responsive AdoMetDC mRNA translation, and we propose that this is achieved by control of ribosomal recognition of the occluded small uORF, either by ribosomal leaky scanning or by programmed -1 frameshifting. In vitro expression demonstrated that both the tiny and the small uORFs are translated. This tiny/small uORF configuration is highly conserved from moss to Arabidopsis thaliana, and a more diverged tiny/small uORF arrangement is found in the AdoMetDC mRNA 5' leader of the single-celled green alga Chlamydomonas reinhardtii, indicating an ancient origin for the uORFs.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Poliaminas Biogénicas/metabolismo , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Secuencia de Bases , Poliaminas Biogénicas/farmacología , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Homeostasis , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
We functionally identified the last remaining step in the plant polyamine biosynthetic pathway by expressing an Arabidopsis thaliana agmatine iminohydrolase cDNA in yeast. Inspection of the whole pathway suggests that the arginine decarboxylase, agmatine iminohydrolase, N-carbamoylputrescine amidohydrolase route to putrescine in plants was inherited from the cyanobacterial ancestor of the chloroplast. However, the rest of the pathway including ornithine decarboxylase and spermidine synthase was probably inherited from bacterial genes present in the original host cell, common ancestor of plants and animals, that acquired the cyanobacterial endosymbiont. An exception is S-adenosylmethionine decarboxylase, which may represent a eukaryote-specific enzyme form.
Asunto(s)
Arabidopsis/enzimología , Cianobacterias/enzimología , Evolución Molecular , Hidrolasas/genética , Putrescina/biosíntesis , Secuencia de Aminoácidos , Arabidopsis/genética , Cloroplastos , Clonación Molecular , Cianobacterias/genética , Etiquetas de Secuencia Expresada , Genes de Plantas , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Poliaminas , Alineación de SecuenciaRESUMEN
S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis. We show that the plant AdoMetDC activity is subject to post-transcriptional control by polyamines. A highly conserved small upstream open reading frame (uORF) in the AdoMetDC mRNA 5' leader is responsible for translational repression of a downstream beta-glucuronidase reporter cistron in transgenic tobacco plants. Elimination of the small uORF from an AdoMetDC cDNA led to increased relative translational efficiency of the AdoMetDC proenzyme in transgenic plants. The resulting increased activity of AdoMetDC caused disruption to polyamine levels with depletion of putrescine, reduction of spermine levels, and a more than 400-fold increase in the level of decarboxylated S-adenosylmethionine. These changes were associated with severe growth and developmental defects. The high level of decarboxylated S-adenosylmethionine was not associated with any change in 5'-methylcytosine content in genomic DNA and S-adenosylmethionine levels were more or less normal, indicating a highly efficient system for maintenance of S-adenosylmethionine levels in plants. This work demonstrates that uORF-mediated translational control of AdoMetDC is essential for polyamine homeostasis and for normal growth and development.