Proteomic analysis of RNA-dependent chromatin association of nuclear proteins
Proteomic analysis of RNA-dependent chromatin association of nuclear proteins
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DOI：
10.1101/391755
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发表时间：
2018-08
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影响因子：
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通讯作者：
Kyoko Hiragami-Hamada;Naoki Tani;J. Nakayama
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作者：
Kyoko Hiragami-Hamada;Naoki Tani;J. Nakayama
已知多种编码和非编码转录本与染色质相关联，且如今越来越多的证据表明，RNA结合蛋白（RBPs）与RNA分子之间的相互作用不仅调控共转录mRNA加工过程，还对细胞核内的其他生物学过程发挥调节作用。尽管通过多种基于质谱的方法已鉴定出超过一千种RBPs，但尚不清楚这些RBPs中哪些实际上与染色质相关联，尤其是通过与RNA的相互作用实现关联的情况。此外，此类RBP - RNA - 染色质相互作用所产生的生物学效应也有待阐明。在此，我们描述了一种基于蛋白质组学的简单方法，用于系统筛选通过RNA分子锚定在染色质和/或不溶性核亚结构上的RBPs。我们使用核糖核酸酶A（RNase A）从染色质组分中释放此类RBPs，并通过质谱分析“RNase A溶解的”蛋白质。利用该方法，我们成功鉴定出156种RNase A溶解的蛋白质，其中144种为已知的RBPs或RBP候选蛋白。有趣的是，非同源末端连接（NHEJ）途径的几个关键参与者在RNase A溶解的组分中富集，并且这些因子通过RNA介导的与染色质的关联似乎依赖于转录延伸。此外，一些参与代谢途径的酶也可通过RNase A处理从染色质和/或不溶性核结构中释放出来。总之，我们的方法通用性强，有可能成为揭示RBP - RNA - 染色质相互作用新生物学功能的有用工具。
Various coding and non-coding transcripts are known to associate with chromatin and now there is accumulating evidence that interaction between RNA-binding proteins (RBPs) and RNA molecules regulate not only co-transcriptional mRNA processing, but also other biological processes within the nucleus. Although over a thousand of RBPs have been identified by several mass spectrometry-based methods, it is still unclear which of these RBPs actually associate with chromatin, especially through interaction with RNAs. In addition, biological outcomes of such RBP-RNA-chromatin interactions are yet to be elucidated. Here we describe a simple proteomics-based method for systematic screening of RBPs that are anchored to chromatin and/or insoluble nuclear substructures by RNA molecules. We used RNase A to release such RBPs from chromatin fraction and analyzed ‘RNase A-solubilized’ proteins by mass spectrometry. Using this method, we were able to identify 156 RNase A-solubilized proteins of which 144 were known RBPs/RBP candidates. Interestingly, several key players of the non-homologous end-joining (NHEJ) pathway were enriched in RNase A-solubilized fraction and the RNA-mediated chromatin association of these factors appeared to be dependent on transcriptional elongation. Furthermore, some enzymes involved in metabolic pathways were also released from chromatin and/or an insoluble nuclear structure by RNase A treatment. In summary, our methodology is highly versatile and is potentially a useful tool to unravel new biological functions for RBP-RNA-chromatin interactions.