RESUMO
Premature termination codons (PTCs) have been shown to initiate degradation of mutant transcripts through the nonsense-mediated messenger RNA (mRNA) decay (NMD) pathway. We report a strategy, termed gene identification by NMD inhibition (GINI), to identify genes harboring nonsense codons that underlie human diseases. In this strategy, the NMD pathway is pharmacologically inhibited in cultured patient cells, resulting in stabilization of nonsense transcripts. To distinguish stabilized nonsense transcripts from background transcripts upregulated by drug treatment, drug-induced expression changes are measured in control and disease cell lines with complementary DNA (cDNA) microarrays. Transcripts are ranked by a nonsense enrichment index (NEI), which relates expression changes for a given transcript in NMD-inhibited control and patient cell lines. The most promising candidates can be selected using information such as map location or biological function; however, an important advantage of the GINI strategy is that a priori information is not essential for disease gene identification. GINI was tested on colon cancer and Sandhoff disease cell lines, which contained previously characterized nonsense mutations in the MutL homolog 1 (MLH1) and hexosaminidase B (HEXB) genes, respectively. A list of genes was produced in which the MLH1 and HEXB genes were among the top 1% of candidates, thus validating the strategy.
Assuntos
Códon sem Sentido , Proteínas de Ligação a DNA , Terminação Traducional da Cadeia Peptídica/genética , RNA Mensageiro , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Transporte , Linhagem Celular , Códon de Terminação/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Hexosaminidase B , Humanos , Masculino , Proteína 1 Homóloga a MutL , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/genética , Proteínas Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/isolamento & purificação , Doença de Sandhoff/tratamento farmacológico , Doença de Sandhoff/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Cadeia beta da beta-Hexosaminidase , beta-N-Acetil-HexosaminidasesRESUMO
Transcripts harboring premature signals for translation termination are recognized and rapidly degraded by eukaryotic cells through a pathway known as nonsense-mediated mRNA decay (NMD). In addition to protecting cells by preventing the translation of potentially deleterious truncated peptides, studies have suggested that NMD plays a broader role in the regulation of the steady-state levels of physiologic transcripts. In Saccharomyces cerevisiae, three trans-acting factors (Upf1p to Upf3p) are required for NMD. Orthologues of Upf1p have been identified in numerous species, showing that the NMD machinery, at least in part, is conserved through evolution. In this study, we demonstrate additional functional conservation of the NMD pathway through the identification of Upf2p homologues in Schizosaccharomyces pombe and humans (rent2). Disruption of S. pombe UPF2 established that this gene is required for NMD in fission yeast. rent2 was demonstrated to interact directly with rent1, a known trans-effector of NMD in mammalian cells. Additionally, fragments of rent2 were shown to possess nuclear targeting activity, although the native protein localizes to the cytoplasmic compartment. Finally, novel functional domains of Upf2p and rent2 with homology to eukaryotic initiation factor 4G (eIF4G) and other translational regulatory proteins were identified. Directed mutations within these so-called eIF4G homology (4GH) domains were sufficient to abolish the function of S. pombe Upf2p. Furthermore, using the two-hybrid system, we obtained evidence for direct interaction between rent2 and human eIF4AI and Sui1, both components of the translation initiation complex. Based on these findings, a novel model in which Upf2p and rent2 effects decreased translation and accelerated decay of nonsense transcripts through competitive interactions with eIF4G-binding partners is proposed.
Assuntos
Códon sem Sentido , Proteínas Fúngicas/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Compartimento Celular , Citoplasma , Fator de Iniciação Eucariótico 4G , Humanos , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Fatores de Iniciação de Peptídeos/metabolismo , Ligação Proteica , RNA Helicases , Estabilidade de RNA , Schizosaccharomyces , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Distribuição TecidualRESUMO
Ripening represents a complex developmental process unique to plants. We are using tomato fruit ripening mutants as tools to understand the regulatory components that control and coordinate the physiological and biochemical changes which collectively confer the ripe phenotype. We have genetically characterized two loci which result in significant inhibition of the ripening process in tomato, ripening-inhibitor (rin), and non-ripening (nor), as a first step toward isolating genes likely to encode key regulators of this developmental process. A combination of pooled-sample mapping as well as classical restriction fragment length polymorphism (RFLP) analysis has permitted the construction of high-density genetic maps for the regions of chromosomes 5 and 10 spanning the rin and nor loci, respectively. To assess the feasibility of initiating a chromosome walk, physical mapping of high molecular weight genomic DNA has been employed to estimate the relationship between physical distance (in kb) and genetic distance (in cM) around the targeted loci. Based on this analysis, the relationship in the region spanning the rin locus is estimated to be 200-300 kb/cM, while the nor locus region ratio is approximately 200 kb/1 cM. Using RFLP markers tightly linked to rin and nor, chromosome walks have been initiated to both loci in a yeast artificial chromosome (YAC) library of tomato genomic DNA. We have isolated and characterized several YAC clones linked to each of the targeted ripening loci and present genetic evidence that at least one YAC clone contains the nor locus.
