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Abstract：
Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity biotinylation method targeting the RNA and proteins constituents. The method that we termed antibody-mediated proximity labelling coupled to mass spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X chromosome in Drosophila. This analysis identified a number of known RNA-binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila. Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein-RNA interactions in biologically diverse domains.
摘要：
真核染色质被组织成功能域，其特征在于不同的蛋白质组组成和特定的核位置。与被脂质膜包围的细胞器相反，不同染色质结构域的组成描述得相当糟糕且高度动态。为了获得对这些领域的分子洞察并探索它们的组成，我们开发了一种针对RNA和蛋白质成分的基于抗体的邻近生物素化方法。我们称为抗体介导的邻近标记偶联质谱（AMPL-MS）的方法不需要表达融合蛋白，因此构成了一种通用且非常灵敏的方法，以表征染色质结构域的组成基于特定特征蛋白或组蛋白修饰。为了证明我们方法的实用性，我们使用AMPL-MS来表征果蝇中色心的分子特征以及包含过度活跃的X染色体的染色体区域。这项分析确定了许多已知的RNA结合蛋白在过度活跃的X和着丝粒附近，支持我们方法的准确性。此外,它使我们能够表征RNA在这些核体形成中的作用。此外，我们的方法鉴定了一组新的与果蝇着丝粒相关的RNA分子.这些新分子的表征表明着丝粒中R环的形成，我们使用果蝇中R环的新型探针进行了验证。一起来看,AMPL-MS提高了邻近连接的选择性和特异性，从而在生物多样性结构域中发现了弱蛋白质-RNA相互作用。
