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Redox regulation of protein translation in eukaryotes.

机译：真核生物中蛋白质翻译的氧化还原调节。

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摘要

Gene expression may be controlled at multiple levels, e.g., through genomic architecture, transcription and translation. In the current work, we focused on regulation of protein synthesis. Historically, the investigation of the regulation of gene expression at the level of translation lagged behind the transcriptional control because of the lack of accessible high-throughput methods. Our research has begun with the finding of the use of alternative non-AUG start codon in thioredoxin-glutathione reductase (TGR), a selenoprotein involved in redox control during male reproduction. The use of this codon, CUG, relies on the Kozak consensus sequence and ribosomal scanning mechanism. However, the CUG serves as an inefficient start codon that allows downstream in-frame initiation, generating two isoforms of the enzyme in vivo and in vitro from the same mRNA. These findings were extended with the use of systemic, proteome-wide approaches, that supported targeted discovery of initiation start sites. For this purpose, a new technology, ribosomal profiling, was employed. It embraced high-throughput sequencing and offered analyses of ribosome occupancy along the mRNA at a single nucleotide resolution. We applied this technique to examine the interplay between transcription and translation under conditions of hydrogen peroxide treatment in Saccharomyces cerevisiae. Oxidative stress elicited by hydrogen peroxide led to a massive and rapid increase in ribosome occupancy of short upstream open reading frames (uORFs), including those with non-AUG translational starts, and N-terminal regions of ORFs that preceded the transcriptional response. In addition, this treatment induced the synthesis of N-terminally extended proteins and elevated stop codon read-through and frameshift events. It also increased ribosome occupancy at the beginning of ORFs and potentially duration of the elongation step. We identified proteins whose synthesis was rapidly regulated by hydrogen peroxide post-transcriptionally; however, for the majority of genes increased protein synthesis followed transcriptional regulation. Nevertheless, a number of proteins were regulated post-transcriptionally even at the 5 min time point. These data defined the landscape of genome-wide regulation of translation in response to hydrogen peroxide and suggested that "potentiation" (co-regulation of the transcript level and translation) is a feature of oxidative stress. Finally, we expanded this research to better define conditions for ribosome profiling, which are broadly applicable for studies on regulation of translation.
机译：可以例如通过基因组结构，转录和翻译在多个水平上控制基因表达。在当前的工作中，我们专注于蛋白质合成的调控。从历史上看，由于缺乏可访问的高通量方法，在翻译水平上对基因表达调控的研究落后于转录控制。我们的研究始于在硫氧还蛋白-谷胱甘肽还原酶（TGR）中使用替代性非AUG起始密码子的发现，TGR是一种在雄性繁殖过程中参与氧化还原控制的硒蛋白。此密码子CUG的使用依赖于Kozak共有序列和核糖体扫描机制。但是，CUG用作低效​​率的起始密码子，允许下游的框内起始，从同一mRNA体内和体外产生两种同工酶。这些发现通过使用系统的，蛋白质组范围的方法得到扩展，该方法支持有针对性地发现起始起始位点。为此，采用了一种新技术，核糖体谱分析。它包括高通量测序，并提供了以单核苷酸分辨率分析沿mRNA的核糖体占有率的信息。我们应用这项技术来检查在酿酒酵母中过氧化氢处理条件下转录和翻译之间的相互作用。过氧化氢引起的氧化应激导致短的上游开放阅读框（uORF）（包括具有非AUG翻译起始位点和在转录响应之前的ORF的N端区域）的核糖体占有率迅速大幅增加。另外，这种处理诱导了N-末端延伸蛋白的合成以及终止密码子通读和移码事件的升高。它也增加了ORF开始时核糖体的占有率，并可能延长了延伸步骤的持续时间。我们鉴定了蛋白质的合成受到转录后过氧化氢的快速调节。但是，对于大多数基因而言，蛋白质合成遵循转录调控。然而，即使在5分钟的时间点，许多蛋白质仍在转录后受到调控。这些数据定义了响应过氧化氢的全基因组翻译调控的格局，并表明“增强”（转录水平和翻译的共同调控）是氧化应激的特征。最后，我们扩展了这项研究，以更好地定义核糖体谱分析的条件，这些条件广泛适用于翻译调控研究。

著录项

  • 作者

    Gerashchenko, Maxim.;

  • 作者单位

    The University of Nebraska - Lincoln.;

  • 授予单位 The University of Nebraska - Lincoln.;
  • 学科 Chemistry Biochemistry.;Biology Genetics.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 100 p.
  • 总页数 100
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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