Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.
Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.
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DOI：
10.1371/journal.pbio.1000238
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发表时间：
2009-11
期刊：
影响因子：
9.8
通讯作者：
Brown PO
中科院分区：
文献类型：
--
作者：
Hendrickson DG;Hogan DJ;McCullough HL;Myers JW;Herschlag D;Ferrell JE;Brown PO
一种特定的microRNA通过对编码它们的mRNA的丰度和翻译的一致影响来减少数百种蛋白质的合成。微小RNA（miRNAs）通过干扰靶mRNA的翻译、稳定性或两者来转录后调节基因表达。我们试图剖析翻译抑制和mRNA衰变对microRNA调控的各自贡献。我们确定了一个特定的miRNA，miR-124的直接目标，凭借他们的协会与Argonaute蛋白，miRNA效应复合物的核心组成部分，在人类组织培养细胞中的miR-124转染响应。与此同时，我们评估mRNA水平，并获得翻译谱使用一种新的全球方法来分析在蔗糖梯度上分离的多核糖体。对这些增殖性人类细胞中108，000个基因的翻译谱的分析表明，翻译的基本特征与以前在快速生长的酿酒酵母中观察到的相似。对于由miR-124特异性募集到Argonaute蛋白的10600个mRNA，我们发现mRNA丰度和推断的翻译速率都在很大的动态范围内降低。这些miR-124靶标的mRNA水平的变化大于翻译的变化，平均分别下降了35%和12%。此外，没有可识别的mRNA靶亚组，其中翻译反应占主导地位。通过miR-124的存在，对于数百个miR-124靶标，核糖体占有率（与核糖体相关的给定基因的转录物的分数）和核糖体密度（每单位长度的编码序列结合的核糖体的平均数目）都选择性地降低。从观察到的mRNA丰度变化和翻译谱推断的蛋白丰度变化与Western分析直接测定的12种蛋白中的11种的变化密切匹配，表明我们的测定捕获了大部分miR-124介导的调控。这些结果表明，miRNA抑制翻译起始或刺激核糖体下降优先接近起始位点，并不符合抑制多肽延伸，或新生多肽降解显着贡献的miRNA介导的调节增殖HEK 293 T细胞。观察到数百种miR-124靶点的mRNA丰度和翻译速率的一致变化与miRNA靶向的这两种调节结果之间的功能联系以及翻译和mRNA衰减之间的充分记录的相互关系一致。人类基因组包含了调控数千个基因表达的时间和幅度的指令。microRNA是重要的调节RNA，其通过与来自这些基因的信使RNA中的互补片段配对来调节数十至数百个特定基因的表达水平，从而降低它们的稳定性和它们翻译成蛋白质。虽然认识到microRNA的重要性，但对同源mRNA的降解或翻译抑制对蛋白质合成的总体影响的相对贡献或这两种调节机制之间的联系知之甚少。我们设计了一种简单、经济的方法来系统地测量mRNA翻译谱，然后将这种方法与基因表达分析相结合，来测量人类microRNA miR-124对其mRNA靶点的丰度和表观翻译速率的影响。我们发现，对于通过与microRNA效应复合物的结合而鉴定的miR-124的10600 mRNA靶点，大约四分之三的估计蛋白质合成减少可以通过mRNA丰度的变化来解释。尽管靶mRNA翻译效率的明显变化幅度较小，但它们与这些RNA丰度的变化高度相关，表明microRNA介导的翻译抑制与mRNA衰变之间存在功能联系。
A specific microRNA reduces the synthesis of hundreds of proteins via concordant effects on the abundance and translation of the mRNAs that encode them. MicroRNAs (miRNAs) regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for ∼8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For ∼600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes) and ribosome density (the average number of ribosomes bound per unit length of coding sequence) were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124–mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the start site and are not consistent with inhibition of polypeptide elongation, or nascent polypeptide degradation contributing significantly to miRNA-mediated regulation in proliferating HEK293T cells. The observation of concordant changes in mRNA abundance and translational rate for hundreds of miR-124 targets is consistent with a functional link between these two regulatory outcomes of miRNA targeting, and the well-documented interrelationship between translation and mRNA decay. The human genome contains directions to regulate the timing and magnitude of expression of its thousands of genes. MicroRNAs are important regulatory RNAs that tune the expression levels of tens to hundreds of specific genes by pairing to complimentary stretches in the messenger RNAs from these genes, thereby reducing their stability and their translation into protein. Although the importance of microRNAs is appreciated, little is known about the relative contributions of degradation or repression of translation of the cognate mRNAs to the overall effects on protein synthesis, or the links between these two regulatory mechanisms. We devised a simple, economical method to systematically measure mRNA translation profiles, then applied this method, in combination with gene expression analysis, to measure the effects of the human microRNA miR-124 on the abundance and apparent translation rate of its mRNA targets. We found that for the ∼600 mRNA targets of miR-124 that were identified by their association with microRNA effector complexes, around three quarters of the reduction in estimated protein synthesis was explained by changes in mRNA abundance. Although the apparent changes in translation efficiencies of the targeted mRNAs were smaller in magnitude, they were highly correlated with changes in the abundance of those RNAs, suggesting a functional link between microRNA-mediated repression of translation and mRNA decay.
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影响因子：
64.8
作者：
Baek, Daehyun;Villen, Judit;Shin, Chanseok;Camargo, Fernando D.;Gygi, Steven P.;Bartel, David P.
通讯作者：
Bartel, David P.
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影响因子：
14.9
作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
4.1
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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