Translation of 5' leaders is pervasive in genes resistant to eIF2 repression.
Translation of 5' leaders is pervasive in genes resistant to eIF2 repression.
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DOI：
10.7554/elife.03971
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发表时间：
2015-01-26
期刊：
影响因子：
7.7
通讯作者：
Baranov PV
中科院分区：
文献类型：
--
作者：
Andreev DE;O'Connor PB;Fahey C;Kenny EM;Terenin IM;Dmitriev SE;Cormican P;Morris DW;Shatsky IN;Baranov PV
真核细胞在各种应激条件下迅速减少蛋白质合成。这可以通过磷酸化介导的关键翻译起始因子真核起始因子2（eIF2）的失活来实现。然而，某些mRNA的持续翻译是部署足够的应激反应所必需的。我们在亚砷酸钠诱导的严重应激条件下对培养的人类细胞进行了核糖体分析。虽然这导致了5.4倍的一般翻译抑制，但某些单个mRNA的蛋白质编码开放阅读框（ORF）表现出对抑制的抗性。几乎所有的抗性转录物都具有至少一个有效翻译的上游开放阅读框（uORF），其在正常条件下抑制主要编码ORF的翻译。两种鉴定的抗胁迫mRNA（PPP1R15 B和IFRD1）的位点特异性诱变证明，在两种情况下，单个uORF足以用于eIF2介导的翻译控制。系统发育分析表明，至少有两个调控uORF（即，在SLC35A4和MIEF1）编码功能蛋白质产物。DOI：http://dx.doi.org/10.7554/eLife.03971.001蛋白质执行活细胞的基本任务，基因包含在其DNA中制造蛋白质的指令。这些指令被复制成mRNA分子，然后被称为核糖体的分子机器读取并翻译mRNA以构建蛋白质。翻译过程的第一步称为“起始”，需要一种名为eIF2的蛋白质与核糖体一起工作。这一步涉及识别一个称为起始密码子的指令，它标志着mRNA编码序列的开始。mRNA分子起始密码子之前的部分通常不被核糖体翻译，因此被称为5′非翻译区。构建蛋白质需要能量和资源，因此它受到精心调控。如果一个细胞受到压力，例如暴露在有害的化学物质中，它就会减少蛋白质的产生，以保护其资源。这种蛋白质生产的下调部分是通过细胞化学修饰其eIF2蛋白质，使它们不太能够启动翻译来实现的。然而，应激细胞仍然继续产生更多的某些蛋白质，帮助它们对抗压力。这些蛋白质的mRNA分子在5′非翻译区至少含有一个其他起始密码子。从这样的起始密码子翻译的序列被称为上游开放阅读框架（或简称uORF），这一特征被认为有助于某些蛋白质在低水平的活性eIF2下仍能表达。Andreev，奥康纳等人现在已经分析了哪些mRNA在人类细胞中被翻译，这些细胞已经用诱导应激的化学物质处理，并使eIF 2蛋白不太能够启动翻译。为了做到这一点，一种称为核糖体分析的技术被用来识别在用这种化学物质处理后不久与核糖体结合的所有mRNA分子。应激细胞中大多数mRNA的总体翻译降低到正常水平的四分之一。然而，Andreev、奥康纳等人观察到，在化学处理后，少数mRNA的翻译几乎与正常情况一样继续，甚至增加。值得注意的是，这些mRNA中的大多数编码调节蛋白，这不是大量需要的。除了一个例外，所有这些抗性mRNA都含有uORF。在未应激的细胞中，这些uORF被有效翻译，而相同mRNA的编码序列被翻译效率较低。Andreev，奥康纳等人认为，这两个特征可用于鉴定当细胞受到应激时仍被翻译成工作蛋白的mRNA。现在需要进一步的工作来探索这些uORF的翻译允许mRNA抵抗压力的机制。DOI：http://dx.doi.org/10.7554/eLife.03971.002网站
Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products. DOI: http://dx.doi.org/10.7554/eLife.03971.001 Proteins carry out essential tasks for living cells and genes contain the instructions to make proteins within their DNA. These instructions are copied to make a molecule of mRNA, and a molecular machine known as a ribosome then reads and translates the mRNA to build the protein. The first step in the translation process is called ‘initiation’ and requires a protein called eIF2 to work together with the ribosome. This step involves identifying an instruction called the start codon that marks the beginning of the mRNA's coding sequence. The section of an mRNA molecule before the start codon is not normally translated by the ribosome and is hence called the 5′ untranslated region. Building proteins requires energy and resources, and so it is carefully regulated. If a cell is stressed, such as by being exposed to harmful chemicals, it makes fewer proteins in order to conserve its resources. This down-regulation of protein production is achieved in part by the cell chemically modifying its eIF2 proteins to make them less able to initiate translation. However, stressed cells still continue to make more of certain proteins that help them to combat stress. The mRNA molecules for some of these proteins contain at least one other start codon in the 5′ untranslated region. The sequence that would be translated from such a start codon is known as an upstream open reading frame (or uORF for short)—and this feature is thought to help certain proteins to still be expressed despite low levels of active eIF2. Andreev, O'Connor et al. have now analysed which mRNAs are translated in human cells that have been treated with a chemical that induces stress and makes the eIF2 protein less able to initiate translation. To do so, a technique called ribosome profiling was used to identify all of the mRNA molecules bound to ribosomes shortly after treatment with this chemical. Overall translation of most mRNAs in stressed cells was reduced to a quarter of the normal level. However, Andreev, O'Connor et al. observed that the translation of a few mRNAs continued almost as normal, or even increased, after the chemical treatment. Notably, most of these mRNAs encoded regulatory proteins, which are not required in large amounts. With one exception, all of these resistant mRNAs contained uORFs. In unstressed cells, these uORFs were efficiently translated, while the same mRNA's coding sequences were translated less efficiently. Andreev, O'Connor et al. suggest that these two features could be used to identify mRNAs that are still translated into working proteins when cells are stressed. Further work is now needed to explore the mechanisms by which translation of these uORFs allows mRNAs to resist the stress. DOI: http://dx.doi.org/10.7554/eLife.03971.002