In vivo interrogation of central nervous system translatome by polyribosome fractionation.
In vivo interrogation of central nervous system translatome by polyribosome fractionation.
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DOI：
10.3791/51255
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发表时间：
2014-04
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影响因子：
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通讯作者：
Wilson Pak-Kin Lou;Avni Baser;Stefan Klussmann;A. Martin-Villalba
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文献类型：
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作者：
Wilson Pak-Kin Lou;Avni Baser;Stefan Klussmann;A. Martin-Villalba
基因表达涉及多个过程，包括转录、翻译和蛋白质的稳定性。这些步骤中的每一步都受到严格的调控，影响着蛋白质丰度的最终动态。在翻译阶段存在各种调节机制，使得单独的mRNA水平不能作为基因表达的可靠指标。此外，信使核糖核酸翻译的局部调控尤其与神经元功能有关，这使得“转译经济学”成为神经生物学关注的焦点。该方法可作为转录组和蛋白质组学之间的桥梁。在这里，我们描述了对多聚核糖体分离技术的基本修改，该技术基于主动翻译的mRNAs与多个核糖体的关联以及它们在蔗糖梯度中的差异沉淀来询问翻译体。传统上，由于材料的数量有限和脂肪组织成分的存在，使用活体样本，特别是中枢神经系统(CNS)样本的工作被证明是具有挑战性的。为了解决这一问题，所描述的方案被特别优化，以使用最少量的中枢神经系统材料，如使用单个小鼠脊髓和大脑所证明的那样。简而言之，提取中枢神经系统组织，并用放线菌酮将翻译的核糖体固定在mRNAs上。然后进行髓磷脂浮选以去除富含脂质的成分。分级是在蔗糖梯度上进行的，其中mRNAs根据它们的核糖体负载而分离。分离的部分适合于一系列下游分析，包括新的全基因组分析技术。
Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting 'translatomics' to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics. Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.