RESUMO
The human oxidative phosphorylation (OxPhos) system consists of approximately 90 proteins encoded by nuclear and mitochondrial genomes and serves as the primary cellular pathway for ATP biosynthesis. While the core protein machinery for OxPhos is well characterized, many of its assembly, maturation, and regulatory factors remain unknown. We exploited the tight transcriptional control of the genes encoding the core OxPhos machinery to identify novel regulators. We developed a computational procedure, which we call expression screening, which integrates information from thousands of microarray data sets in a principled manner to identify genes that are consistently co-expressed with a target pathway across biological contexts. We applied expression screening to predict dozens of novel regulators of OxPhos. For two candidate genes, CHCHD2 and SLIRP, we show that silencing with RNAi results in destabilization of OxPhos complexes and a marked loss of OxPhos enzymatic activity. Moreover, we show that SLIRP plays an essential role in maintaining mitochondrial-localized mRNA transcripts that encode OxPhos protein subunits. Our findings provide a catalogue of potential novel OxPhos regulators that advance our understanding of the coordination between nuclear and mitochondrial genomes for the regulation of cellular energy metabolism.
Assuntos
Biologia Computacional/métodos , Homeostase , Mitocôndrias/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Fosforilação Oxidativa , RNA/química , RNA/genética , RNA Mitocondrial , Proteínas de Ligação a RNA/genéticaRESUMO
We determined the physical locations of the heterodimeric Ecdysone receptor/Ultraspiracle (ECR/USP) nuclear hormone receptor complex throughout the entire nonrepetitive genome of Drosophila melanogaster using a cell line (Kc167) that differentiates in response to 20-hydroxyecdysone (20-HE). 20-HE, the natural ligand of this complex, controls major aspects of insect development, including molting, metamorphosis, and reproduction. Direct gene targets of 20-HE signaling were identified by combining this physical binding-site profiling with gene expression profiling after treatment with 20-HE. We found 502 significant regions of ECR/USP binding throughout the genome. Only 42% of these regions are nearby genes that are 20-HE responsive in these cells. However, at least three quarters of the remaining ECR/USP regions are near 20-HE-regulated genes in other tissue and cell types during metamorphosis, suggesting that binding at many regulatory elements in the genome is largely noncell-type specific. The majority (21/26) of the early targets of 20-HE encode transcriptional regulatory factors. To determine whether any of these targets are required for the morphological differentiation of these cells, we used RNAi to reduce the expression of each of the 26 early genes. Accordingly, we found that three direct targets of ECR/USP--hairy, vrille, and Hr4--are required for cellular differentiation in response to the hormone. Initial mutational analysis of vrille in vivo reveals that it is required for metamorphosis.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Genoma/genética , Receptores de Esteroides/genética , Fatores de Transcrição/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Mapeamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ecdisterona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
We used a maskless photolithography method to produce DNA oligonucleotide microarrays with unique probe sequences tiled throughout the genome of Drosophila melanogaster and across predicted splice junctions. RNA expression of protein coding and nonprotein coding sequences was determined for each major stage of the life cycle, including adult males and females. We detected transcriptional activity for 93% of annotated genes and RNA expression for 41% of the probes in intronic and intergenic sequences. Comparison to genome-wide RNA interference data and to gene annotations revealed distinguishable levels of expression for different classes of genes and higher levels of expression for genes with essential cellular functions. Differential splicing was observed in about 40% of predicted genes, and 5440 previously unknown splice forms were detected. Genes within conserved regions of synteny with D. pseudoobscura had highly correlated expression; these regions ranged in length from 10 to 900 kilobase pairs. The expressed intergenic and intronic sequences are more likely to be evolutionarily conserved than nonexpressed ones, and about 15% of them appear to be developmentally regulated. Our results provide a draft expression map for the entire nonrepetitive genome, which reveals a much more extensive and diverse set of expressed sequences than was previously predicted.
