Advancing the functional utility of PAR-CLIP by quantifying background binding to mRNAs and lncRNAs.
Advancing the functional utility of PAR-CLIP by quantifying background binding to mRNAs and lncRNAs.
复制标题
通过量化与mRNA和LNCRNA的背景结合来推进PAR-CLIP的功能效用。
DOI：
10.1186/gb-2014-15-1-r2
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发表时间：
2014-01-07
期刊：
影响因子：
12.3
通讯作者：
Keene JD
中科院分区：
文献类型：
--
作者：
Friedersdorf MB;Keene JD
序列特异性RNA结合蛋白是基因表达的重要调节因子。最近开发了几种相关的基于交联的高通量测序方法，包括PAR-CLIP，以确定全局蛋白质-RNA相互作用的直接结合位点。然而，没有研究定量地解决了背景结合这些方法产生的数据集的贡献。我们测量了PAR-CLIP数据中的非特异性RNA背景，证明共价交联背景结合在实验室中是常见的、可重复的和明显通用的。我们表明，背景的定量测定是必不可少的，以确定大多数RNA结合蛋白的目标，并可以大大提高基序分析。我们还表明，通过应用背景校正未知的结合特异性的RNA结合蛋白，Caprin 1，我们可以确定一个以前未识别的RNA识别元件，否则在PAR-CLIP研究中不明显。整体RNA-蛋白质交联的经验背景测量是其他实验对照的必要补充，例如进行重复，因为共价交联的背景信号是可重现的，否则是不可避免的。识别和量化背景的贡献扩展了PAR-CLIP的实用性，并且可以提高对蛋白质-RNA特异性、蛋白质-RNA亲和力和蛋白质-RNA缔合动力学的机制理解。
Sequence specific RNA binding proteins are important regulators of gene expression. Several related crosslinking-based, high-throughput sequencing methods, including PAR-CLIP, have recently been developed to determine direct binding sites of global protein-RNA interactions. However, no studies have quantitatively addressed the contribution of background binding to datasets produced by these methods. We measured non-specific RNA background in PAR-CLIP data, demonstrating that covalently crosslinked background binding is common, reproducible and apparently universal among laboratories. We show that quantitative determination of background is essential for identifying targets of most RNA-binding proteins and can substantially improve motif analysis. We also demonstrate that by applying background correction to an RNA binding protein of unknown binding specificity, Caprin1, we can identify a previously unrecognized RNA recognition element not otherwise apparent in a PAR-CLIP study. Empirical background measurements of global RNA-protein crosslinking are a necessary addendum to other experimental controls, such as performing replicates, because covalently crosslinked background signals are reproducible and otherwise unavoidable. Recognizing and quantifying the contribution of background extends the utility of PAR-CLIP and can improve mechanistic understanding of protein-RNA specificity, protein-RNA affinity and protein-RNA association dynamics.
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