RESUMO
MOTIVATION: The COP9 signalosome is a highly conserved multi-protein complex consisting of eight subunits, which influences key developmental pathways through its regulation of protein stability and transcription. In Arabidopsis thaliana, mutations in the COP9 signalosome exhibit a number of diverse pleiotropic phenotypes. Total or partial loss of COP9 signalosome function in Arabidopsis leads to misregulation of a number of genes involved in DNA methylation, suggesting that part of the pleiotropic phenotype is due to global effects on DNA methylation. RESULTS: We determined and analyzed the methylomes and transcriptomes of both partial- and total-loss-of-function Arabidopsis mutants of the COP9 signalosome. Our results support the hypothesis that the COP9 signalosome has a global genome-wide effect on methylation and that this effect is at least partially encoded in the DNA. Our analyses suggest that COP9 signalosome-dependent methylation is related to gene expression regulation in various ways. Differentially methylated regions tend to be closer in the 3D conformation of the genome to differentially expressed genes. These results suggest that the COP9 signalosome has a more comprehensive effect on gene expression than thought before, and this is partially related to regulation of methylation. The high level of COP9 signalosome conservation among eukaryotes may also suggest that COP9 signalosome regulates methylation not only in plants but also in other eukaryotes, including humans. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis , Complexo do Signalossomo COP9 , Epigênese GenéticaRESUMO
The roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits are largely unclear, although some are essential, while others are thought to have regulatory roles. The 'e' subunit, also known as Int-6/Int6, is a candidate for a regulatory subunit as it is not essential for translation initiation in yeasts. eIF3e associates with the COP9 signalosome, and localizes to the nucleus in certain tissues. To further elucidate the roles of eIF3e, we have taken a genetic approach using Arabidopsis as a model system. Overexpression of eIF3e results in defects similar to mutations in the COP9 signalosome. eIF3e protein, but not transcript, over accumulates in csn mutants, and eIF3e is degraded in a proteasome-dependent fashion. In vitro and in vivo assays suggest that excess eIF3e inhibits translation. We conclude that the COP9 signalosome maintains a precise regulation of eIF3e levels, which is necessary for normal development in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/biossíntese , Arabidopsis/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Transcrição GênicaRESUMO
The roles of individual Eukaryotic translation Initiation Factor 3 (eIF3) subunits are largely unclear. Though some are essential, while others are thought to have regulatory roles. The "e" subunit, also known as Int-6, is a candidate for a regulatory subunit as it is not essential for translation initiation in yeasts. To further elucidate the roles of eIF3e, we have employed an interaction-trap screen using the yeast two-hybrid system. eIF3e interacts in yeast with subunits of the ribosome, COP9 signalosome and 26S proteasome. These interactions mesh well with our recent results which showed that eIF3e is degraded in a CSN-dependent, proteasome-dependent fashion, and inhibits translation when present in excess.
RESUMO
Eukaryotic translation initiation factor 3 (eIF3) consists of core subunits that are conserved from yeast to man as well as less conserved, noncore, subunits with potential regulatory roles. Whereas core subunits tend to be indispensable for cell growth, the roles of the noncore subunits remain poorly understood. We addressed the hypothesis that eIF3 noncore subunits have accessory functions that help to regulate translation initiation, by focusing on the Arabidopsis thaliana eIF3h subunit. Indeed, eIF3h was not essential for general protein translation. However, results from transient expression assays and polysome fractionation indicated that the translation efficiency of specific 5' mRNA leader sequences was compromised in an eif3h mutant, including the mRNA for the basic domain leucine zipper (bZip) transcription factor ATB2/AtbZip11, translation of which is regulated by sucrose. Among other pleiotropic developmental defects, the eif3h mutant required exogenous sugar to transit from seedling to vegetative development, but it was hypersensitive to elevated levels of exogenous sugars. The ATB2 mRNA was rendered sensitive to the eIF3h level by a series of upstream open reading frames. Moreover, eIF3h could physically interact with subunits of the COP9 signalosome, a protein complex implicated primarily in the regulation of protein ubiquitination, supporting a direct biochemical connection between translation initiation and protein turnover. Together, these data implicate eIF3 in mRNA-associated translation initiation events, such as scanning, start codon recognition, or reinitiation and suggest that poor translation initiation of specific mRNAs contributes to the pleiotropic spectrum of phenotypic defects in the eif3h mutant.
Assuntos
Regiões 5' não Traduzidas/genética , Arabidopsis/genética , Fator de Iniciação 3 em Eucariotos/genética , Regulação da Expressão Gênica de Plantas/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica , Complexo do Signalossomo COP9 , Análise Mutacional de DNA , Fator de Iniciação 3 em Eucariotos/biossíntese , Fatores de Iniciação em Eucariotos/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Complexos Multiproteicos , Mutação/genética , Peptídeo Hidrolases , Subunidades Proteicas/biossíntese , Subunidades Proteicas/genética , Proteínas/genética , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.