RESUMO
Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm5U34), 5-methoxycarbonylmethyluridine (mcm5U34), and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s²U34) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA(Lys)(s²UUU), tRNA(Gln)(s²UUG), and tRNA(Glu)(s²UUC), which in a wild-type background contain the mcm5s²U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U34. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm5s²U nucleoside in tRNA(Lys)(mcm5s²UUU), tRNA(Gln)(mcm5s²UUG), and tRNA(Glu)(mcm5s²UUC). These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U34 are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.
Assuntos
RNA de Transferência de Glutamina/genética , RNA de Transferência de Ácido Glutâmico/genética , RNA de Transferência de Lisina/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA/genética , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Biossíntese de Proteínas , RNA de Transferência/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Telômero/genéticaRESUMO
We have developed a multistep strategy that integrates data from several large-scale experiments that suffer from systematic between-experiment variation. This strategy removes such variation that would otherwise mask differences of interest. It was applied to the evaluation of wood chemical analysis of 736 hybrid aspen trees: wild-type controls and transgenic trees potentially involved in wood formation. The trees were grown in four different greenhouse experiments imposing significant variation between experiments. Pyrolysis coupled to gas chromatography/mass spectrometry (Py-GC/MS) was used as a high throughput-screening platform for fingerprinting of wood chemotype. Our proposed strategy includes quality control, outlier detection, gene specific classification, and consensus analysis. The orthogonal projections to latent structures discriminant analysis (OPLS-DA) method was used to generate the consensus chemotype profiles for each transgenic line. These were thereafter compiled to generate a global dataset. Multivariate analysis and cluster analysis techniques revealed a drastic reduction in between-experiment variation that enabled a global analysis of all transgenic lines from the four independent experiments. Information from in-depth analysis of specific transgenic lines and independent peak identification validated our proposed strategy.
Assuntos
Plantas Geneticamente Modificadas/genética , Populus/genética , Árvores/genética , Madeira/genética , Análise por Conglomerados , Análise Discriminante , Cromatografia Gasosa-Espectrometria de Massas , Modelos Estatísticos , Análise Multivariada , Fenótipo , Plantas Geneticamente Modificadas/química , Populus/química , Análise de Componente Principal , Árvores/química , Madeira/químicaRESUMO
A strategy for optimizing LC-MS metabolomics data processing is proposed. We applied this strategy on the XCMS open source package written in R on both human and plant biology data. The strategy is a sequential design of experiments (DoE) based on a dilution series from a pooled sample and a measure of correlation between diluted concentrations and integrated peak areas. The reliability index metric, used to define peak quality, simultaneously favors reliable peaks and disfavors unreliable peaks using a weighted ratio between peaks with high and low response linearity. DoE optimization resulted in the case studies in more than 57% improvement in the reliability index compared to the use of the default settings. The proposed strategy can be applied to any other data processing software involving parameters to be tuned, e.g., MZmine 2. It can also be fully automated and used as a module in a complete metabolomics data processing pipeline.
Assuntos
Cromatografia Líquida , Espectrometria de Massas , Metabolômica , Projetos de Pesquisa , Adulto , Humanos , Masculino , Plantas/metabolismo , UrináliseRESUMO
In metabolomics studies there is a clear increase of data. This indicates the necessity of both having a battery of suitable analysis methods and validation procedures able to handle large amounts of data. In this review, an overview of the metabolomics data processing pipeline is presented. A selection of recently developed and most cited data processing methods is discussed. In addition, commonly used chemometric and machine learning analysis methods as well as validation approaches are described.