TRAP-RC，翻译核糖体亲和纯化的稀有细胞群<em>果蝇</em>胚胎
Summary
Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.
Abstract
Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.
Introduction
了解发育过程中细胞如何获得特殊性能是至关重要的，以欣赏器官的复杂性，以及他们对病理状况的潜在变化。有一个大的研究推动破译的基因调控网络，通过unrevealing全球基因表达谱underly细胞规范和分化，聚合酶II绑定状态，在调控序列和组蛋白翻译后修饰的转录因子占用。
为了评估基因表达在全球范围内芯片和RNA测序方法的使用。微阵列容许检测mRNA的丰度，而核糖核酸-SEQ是更好地面向通知上的不同类型的RNA包括编码和非编码像微RNA，lncRNAs或snRNAs的RNA。然而，这些技术不能区分积极转录，稳态mRNA的，积极-mRNA翻译，和mRNA进入降解过程。测量稳定ST吃了mRNA水平是已知的蛋白质成分1-3的不良估计。在相反，识别积极-mRNA翻译给出蛋白产生的更精确的图像。
然而，转录研究主要被引导在整个生物体水平通过比较增益或的特定因子的功能丧失与野生型条件4-7或解剖组织，使得基因表达谱难以解释。在细胞靶向工具，亲和纯化和灵敏度的改进的最新进展现在允许执行的全基因组分析在活生物体小细胞群体或甚至单个细胞。
在果蝇中，转基因株系大型集合经由GAL4 / UAS系统8-10使不同的组织和细胞亚群的具体时间和空间定向，得到所需的细胞功能的分子机制的更准确的定量。
<p cl屁股="“jove_content”">在新开发的方法多聚核糖体分析分馏被描述为一种捕获积极-RNA翻译11。基于蔗糖梯度离心该方法允许多核糖体级分和测定mRNA的的选择翻译的全基因组时与微阵列或深度测序进行分析。多核糖体隔离可与核酸酶消化，以确定对应于在翻译过程12由核糖体保护RNA片段核糖体的脚印。核糖体足迹增加核糖核酸翻译和RNA序列级别分辨率和核糖体定位定量的准确性，使得能够测量翻译的速率。但是，到目前为止还没有被应用到translatomes的小区固有的隔离，其主要原因是在核糖体足迹过程结束时获得的RNA产量的急剧下降。鉴于RNA的大小选择可产生潜在的错误 – 对从也可以以类似的方式保护不同的RNA的RNP ositives，需要进一步的发展以提高核糖体足迹并使其适应小区特有的方法。这种方法之一，被称为翻译核糖体亲和纯化（TRAP）已在2008年已描述由海曼等人 。并应用于从小鼠的神经元的一个子集隔离translatome。通过以细胞类型特异性的方式表位标记核糖体蛋白质，核糖可以选择性地纯化无细胞解离和排序的费力的工序。在这种情况下，60S核糖体蛋白L10A在核糖体的表面，具有标记的eGFP 13。自2008年以来，一些研究已经用这种方法在不同的物种，从小鼠14-19 为 X. 蟾 20,21，斑马鱼22,23和拟南芥24。该陷阱的方法也已经适应了果蝇模型，并使用普里FY细胞类型特异性从神经元细胞25和星形胶质细胞26的mRNA。多功能二进制GAL4 / UAS系统被用来表示在一个组织特异性方式GFP标记RpL10A。成蝇首脑进行解剖，以执行从（由泛神经ELAV-Gal4的驱动程序有针对性的）神经细胞和孤立的RNA核糖体的选择进行了测序。大量的上调基因对应于那些已知待表达在神经系统，这表明该方法具有良好的特异性和促使我们以使其适应稀有细胞群在显影果蝇胚胎本（小于1细胞的％） 。
在果蝇模型中，胚胎阶段是首选发育过程的研究模型，作为分子的行动者和形态发生运动是进化上保守。在这个模型中迄今开展的唯一的组织特异性转录的方法使用电池或核这样已执行rting并只能够研究稳态转录27-31，创造一个需要专用于组织/细胞特异性translatome分析在果蝇胚胎的方法。在这里，我们报告奉献给果蝇胚胎的第一个陷阱协议。用这种方法，从事合mRNA翻译在每个胚胎周围100肌细胞的一个非常有限的细胞群被成功分离高质量和特异性。二进制GAL4 / UAS系统用于驱动在肌肉的亚群GFP标记RpL10A的表达无任何毒性，没有明显的表型或发育迟缓如前所示25。 果蝇胚胎具有每hemisegment 30的肌肉，这将引起肌肉组织幼虫。通过使用发现的懒散身份基因的附近的调节区，6出来的30多成核的肌肉的有针对性的。在该测定中，核糖体使用的是翻译延伸固定在mRNA上抑制剂放线菌酮。胞质提取物随后用于亲和纯化用GFP抗体 – 包被的磁珠。纯化的RNA质量利用生物分析仪进行了验证。定量逆转录PCR（RT-qPCR的）被用来确定特异性和mRNA分离灵敏度和证明我们的优化的协议是高效率的。
Protocol
Representative Results
Discussion
本文介绍一种改性的翻译核糖体亲和纯化协议（称为“陷阱-RC”）致力于在果蝇胚胎罕见的细胞群的研究。信息被设置在用于特定核糖核酸成功分离适于微阵列或RNA-SEQ分析，所需的生物材料，即 ，1）的数量和优化过程为胚胎裂解和多核糖体提取的关键步骤，收率2）珠/抗体对裂解物的比率为最佳免疫沉淀; 3）步骤允许减少的背景，包括洗涤缓冲液组合物，以便运行的TRAP的rc实验以高特异性和灵敏度。
这种协议产生再生的数据使得它容易地应用到任何其他类型的细胞。这种技术使得可以分析差异基因表达在积极-翻译mRNA的水平，从而铺路理解在一个规范表达 IFIC细胞类型在一个特定的时间窗口。但是请注意，蛋白丰度将取决于翻译和退化过程的速率。为TRAP方法的主要限制是它不能测量蛋白质含量以精确定量的方式或以检测翻译后修饰。通过测量沿mRNA的核糖体体密度提高量化，并且在这样做时，在一个细胞类型特异性的方式可以使TRAP结合核糖体足迹一个非常强大的工具。在最近的论文34，在人胚胎肾293细胞中进行这种组合。作者跑核酸酶足迹接着RpL10A的使用链亲和素珠诱导型生物素化形式的亲和纯化。这是一个原则证明，有可能在整个有机体而靶向特定的细胞类型，所述的限制是需要的目的生物物质的量进行核糖体足迹。
“>表演TRAP实验在具有全局转录分析并行将使得有可能给新转录和翻译actively-的RNA。这将是深深信息发生在特定的发育上下文或特定细胞群的潜在转录后机制之间跟踪比例 – 用微RNA的例子。总之，TRAP是一种高效，特异性和灵敏的用于识别的RNA结合到在细胞特异性方式积极-翻译核糖体方法。这种方法可以在各种生物和组织类型的使用。这里所描述的新的TRAP的rc协议稀有细胞群（总细胞数小于1％）和最终材料的足够用于在全基因组水平的后续分析的量进行了优化。
这种方法的一个可能限制是一个强有力的司机的要求，以产生标记的核糖体在足够量的小样ETE与内源的未标记的人在靶细胞类型。在最近的一项研究25，作者估计10-30％的标记与未标记的核糖体的比例。
为了克服这个问题增加UAS-RpL10A-EGFP拷贝数应大大改进这种平衡。备选地，添加到所描述的遗传背景一个UAS-GAL4基因将放大生产GAL4蛋白质的和间接增加RpL10A-EGFP的表达。这应该有利于更好地占用mRNA上标记的核糖体。
注意，这已经有效的方法，可以更有力通过适应互补的方法带来了更多的定量方面或以发现和解开，这对基因表达的控制至关重要的分子机制作出。
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors thank Nicolas Allegre, Maud Peyny and Jean-Philippe Da Ponte for their excellent technical assistance. This work was supported by the Agence Nationale de la Recherche (ANR-JC-CARDIAC-SPE), the INSERM starting grant, ANR ID-CELL-SPE grant, Equipe FRM and the AFM 15845 grant.
Materials
| HEPES | Sigma | H4034-1006 | |
| 1,2-diheptanoyl-sn-glycero-3-phosphocholine | Avanti Polar Lipids | 850306P | |
| Glycogen | thermo scientific | R0561 | |
| Purified BSA 100X | Biolabs | B9001 | |
| Yeast tRNA | Sigma | R5636 | |
| Super RNase In | Ambion | AM2694 | |
| Antibody GFP clone N86/38 | UC DAVIS/NIH Neuromab Faculty Antibodies Incorporated | 75-132 | |
| Nonidet P40 | Roche | 11754595001 | |
| Precellys 24 Lysis homogenisation | Bertin Technology | ||
| Nuclease free water | Nalgene | ||
| Dynabeads ProteinG | Invitrogen | 10004D | |
| Potassium chloride (KCl) | Applied Biosystem | AM96406 | |
| Magnesium Chloride (MgCl2) | Applied Biosystem | AM95306 | |
| Cellulose waddin unbleached | VWR | 115-2597 | |
| Sieves 112, 355, 710 um | Retsch | ||
| TRiZol | Invitrogen | 15596-018 | |
| MagRack 6 | GE HealthCare | 28948964 | |
| Low binding filter tips | ClearLine | ||
| Rnase free tube 1,5 mL | ClearLine | 390689DD | |
| Tween 20 | Fischer Bioreagent | BP337-500 | |
| Cycloheximide | Sigma | C1985 | |
| Protease inhibitor tablets, Mini-Complete, EDTA-free | Roche | 11836170001 |
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