Lou Wilson Pak-Kin, Baser Avni, Klußmann Stefan, Martin-Villalba Ana
Department of Molecular Neurobiology, German Cancer Research Center (DKFZ).
Department of Molecular Neurobiology, German Cancer Research Center (DKFZ);
J Vis Exp. 2014 Apr 30(86):51255. doi: 10.3791/51255.
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.
基因表达涉及多个过程，包括转录、翻译以及mRNA和蛋白质的稳定性。这些步骤中的每一步都受到严格调控，影响着蛋白质丰度的最终动态变化。在翻译步骤存在多种调控机制，使得仅mRNA水平成为基因表达的不可靠指标。此外，mRNA翻译的局部调控尤其与神经元功能相关，这使得“翻译组学”成为神经生物学关注的焦点。所提出的方法可用于连接转录组学和蛋白质组学。在这里，我们描述了对多核糖体分级分离技术的重要改进，该技术基于活跃翻译的mRNA与多个核糖体的结合及其在蔗糖梯度中的差异沉降来研究翻译组。传统上，处理体内样本，尤其是中枢神经系统（CNS）的样本，由于材料数量有限以及存在脂肪组织成分，已证明具有挑战性。为了解决这个问题，所描述的方案专门针对使用最少数量的CNS材料进行了优化，如使用单只小鼠的脊髓和脑所证明的那样。简要地说，提取CNS组织，并用环己酰亚胺将正在翻译的核糖体固定在mRNA上。然后进行髓磷脂浮选以去除富含脂质的成分。在蔗糖梯度上进行分级分离，其中mRNA根据其核糖体负载进行分离。分离的级分适用于一系列下游分析，包括新的全基因组分析技术。
