RESUMO
DNA damage in Arabidopsis thaliana seedlings results in upregulation of hundreds of genes. One of the earliest and highest levels of induction is displayed by a previously uncharacterized gene that we have termed X-ray induced 1 (XRI1). Analysis of plants carrying a null xri1 allele revealed two distinct requirements for this gene in plant fertility. XRI1 was important for the post-meiotic stages of pollen development, leading to inviability of xri(-) pollen and abnormal segregation of the mutant allele in heterozygous xri1(+/-) plants. In addition, XRI1 was essential for male and female meiosis, as indicated by the complete sterility of homozygous xri1 mutants due to extensive chromosome fragmentation visible in meiocytes. Abolition of programmed DNA double-strand breaks in a spo11-1 mutant background failed to rescue the DNA fragmentation of xri1 mutants, suggesting that XRI1 functions at an earlier stage than SPO11-1 does. Yeast two-hybrid studies identified an interaction between XRI1 and a novel component of the Arabidopsis MND1/AHP2 complex, indicating possible requirements for XRI1 in meiotic DNA repair.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Gametogênese/genética , Meiose/genética , Alelos , Sequência de Aminoácidos , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efeitos da radiação , Clonagem Molecular , Quebras de DNA de Cadeia Dupla , Fragmentação do DNA , Reparo do DNA , DNA de Plantas/genética , Flores/crescimento & desenvolvimento , Meiose/efeitos da radiação , Dados de Sequência Molecular , Alinhamento de Sequência , Raios XRESUMO
Obligatory homologous recombination (HR) is required for chiasma formation and chromosome segregation in meiosis I. Meiotic HR is initiated by DNA double-strand breaks (DSBs), generated by Spo11, a homologue of the archaebacterial topoisomerase subunit Top6A. In Saccharomyces cerevisiae, Rad50, Mre11 and Com1/Sae2 are essential to process an intermediate of the cleavage reaction consisting of Spo11 covalently linked to the 5' termini of DNA. While Rad50 and Mre11 also confer genome stability to vegetative cells and are well conserved in evolution, Com1/Sae2 was believed to be fungal-specific. Here, we identify COM1/SAE2 homologues in all eukaryotic kingdoms. Arabidopsis thaliana Com1/Sae2 mutants are sterile, accumulate AtSPO11-1 during meiotic prophase and fail to form AtRAd51 foci despite the presence of unrepaired DSBs. Furthermore, DNA fragmentation in AtCom1 is suppressed by eliminating AtSPO11-1. In addition, AtCOM1 is specifically required for mitomycin C resistance. Interestingly, we identified CtIP, an essential protein interacting with the DNA repair machinery, as the mammalian homologue of Com1/Sae2, with important implications for the molecular role of CtIP.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Genes de Plantas , Meiose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA , Fragmentação do DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Endonucleases , Humanos , Mitomicina/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Inibidores da Síntese de Ácido Nucleico/metabolismo , Fenótipo , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
During meiosis, homologous chromosomes recognize each other, align, and exchange genetic information. This process requires the action of RecA-related proteins Rad51 and Dmc1 to catalyze DNA strand exchanges. The Mnd1-Hop2 complex has been shown to assist in Dmc1-dependent processes. Furthermore, higher eukaryotes possess additional RecA-related proteins, like XRCC3, which are involved in meiotic recombination. However, little is known about the functional interplay between these proteins during meiosis. We investigated the functional relationship between AtMND1, AtDMC1, AtRAD51, and AtXRCC3 during meiosis in Arabidopsis thaliana. We demonstrate the localization of AtMND1 to meiotic chromosomes, even in the absence of recombination, and show that AtMND1 loading depends exclusively on AHP2, the Arabidopsis Hop2 homolog. We provide evidence of genetic interaction between AtMND1, AtDMC1, AtRAD51, and AtXRCC3. In vitro assays suggest that this functional link is due to direct interaction of the AtMND1-AHP2 complex with AtRAD51 and AtDMC1. We show that AtDMC1 foci accumulate in the Atmnd1 mutant, but are reduced in number in Atrad51 and Atxrcc3 mutants. This study provides the first insights into the functional differences of AtRAD51 and AtXRCC3 during meiosis, demonstrating that AtXRCC3 is dispensable for AtDMC1 focus formation in an Atmnd1 mutant background, whereas AtRAD51 is not. These results clarify the functional interactions between key players in the strand exchange processes during meiotic recombination. Furthermore, they highlight a direct interaction between MND1 and RAD51 and show a functional divergence between RAD51 and XRCC3.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Meiose , Fosfotransferases/genética , Recombinases Rec A/genética , Cromatina/metabolismo , Dimerização , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Vetores Genéticos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Recombinação Genética , Análise de Sequência de DNARESUMO
Translation initiation of eukaryotic mRNAs generally occurs by cap-dependent ribosome scanning. However, certain mRNAs contain internal ribosome entry sites (IRES) allowing cap-independent translation. Several of these IRES-competent transcripts and their corresponding proteins are involved in tumourigenesis. This study focused on IRES-driven translation control during the epithelial to mesenchymal transition (EMT) of hepatocytes that reflects crucial aspects of carcinoma progression. Expression profiling of EMT revealed Laminin B1 (LamB1) to be translationally upregulated. The 5'-untranslated region (UTR) of LamB1 was potent to direct IRES-dependent mRNA utilization of a bicistronic reporter construct. Stringent assays for cryptic promoter and splice sites showed no aberrantly expressed transcripts, suggesting that the reporter activity provided by the leader region of LamB1 mRNA exclusively depends on IRES. In accordance, LamB1 expression increased upon negative interference with cap-dependent translation by expression of human rhinovirus 2A protease or heat shock of cells. Finally, the enhanced expression of LamB1 during EMT correlated with an elevated IRES activity. Together, these data provide first evidence that the 5'-UTR of LamB1 contains a bona fide IRES that directs translational upregulation of LamB1 during stress conditions and neoplastic progression of hepatocytes.
Assuntos
Regiões 5' não Traduzidas/química , Hepatócitos/metabolismo , Laminina/genética , Iniciação Traducional da Cadeia Peptídica , Animais , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Epitélio/metabolismo , Genes Reporter , Resposta ao Choque Térmico , Hepatócitos/citologia , Humanos , Laminina/biossíntese , Mesoderma/metabolismo , Camundongos , Regiões Promotoras Genéticas , Capuzes de RNA/metabolismo , Sítios de Splice de RNA , Regulação para Cima , Proteínas Virais/metabolismoRESUMO
Mnd1 has recently been identified in yeast as a key player in meiotic recombination. Here we describe the identification and functional characterisation of the Arabidopsis homologue, AtMND1, which is essential for male and female meiosis and thus for plant fertility. Although axial elements are formed normally, sister chromatid cohesion is established and recombination initiation appears to be unaffected in mutant plants, chromosomes do not synapse. During meiotic progression, a mass of entangled chromosomes, interconnected by chromatin bridges, and severe chromosome fragmentation are observed. These defects depend on the presence of SPO11-1, a protein that initiates recombination by catalysing DNA double-strand break (DSB) formation. Furthermore, we demonstrate that the AtMND1 protein interacts with AHP2, the Arabidopsis protein closely related to budding yeast Hop2. These data demonstrate that AtMND1 plays a key role in homologous synapsis and in DSB repair during meiotic recombination.