采用绝对定量法的 RNA-蛋白质相互作用的 easyCLIP 分析。
easyCLIP analysis of RNA-protein interactions incorporating absolute quantification.
机构信息
Program in Epithelial Biology, Stanford University, Stanford, CA, USA.
Stanford Program in Cancer Biology, Stanford University, Stanford, CA, USA.
出版信息
Nat Commun. 2021 Mar 10;12(1):1569. doi: 10.1038/s41467-021-21623-4.
Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.
目前缺乏用于鉴定 RNA 结合蛋白 (RBP) 的定量标准，也缺乏用于定义 RBP 靶 RNA 的标准。在这里，我们开发了一种紫外线 (UV) 交联免疫沉淀 (CLIP)-测序方法，即 easyCLIP。easyCLIP 提供了绝对交联率，并且在测序文库制备过程中提高了简单性、效率和可视化 RNA 文库的能力。在 >35 种蛋白质中测量了 >200 个独立的交联实验，确定了一个 RNA 交联率阈值，该阈值可将 RBP 与非 RBP 区分开来，并将靶 RNA 定义为具有复杂频率的 RNA，不太可能是随机蛋白。我们将 easyCLIP 应用于 28 个 RBP 中 28 个最常见的癌症突变，发现 KHDRBS2 R168C、A1CF E34K 和 PCBP1 L100P/Q 癌症突变的每个 RBP 分子的 RNA 结合增加。因此，定量 RBP-RNA 相互作用可以将蛋白质鉴定为 RBP，并确定特定与疾病相关的 RBP 突变对 RNA 结合的影响。
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