UV-crosslinking has proven to be an invaluable tool for the identification of RNA-protein interactomes. The paucity of methods for distinguishing background from bona fide RNA-protein interactions however makes attribution of RNA binding function on UV-crosslinking alone challenging. To address this need, we previously reported an RNA binding protein (RBP) confidence scoring metric, (RCS), incorporating both signal-to-noise (S:N) and protein abundance determinations to distinguish high and low confidence candidate RBPs. Although RCS has utility, we sought a direct metric for quantification and comparative evaluation of protein-RNA interactions. Here we propose the use of protein-specific UV-crosslinking efficiency (%CL), representing the molar fraction of a protein that is crosslinked to RNA, for functional evaluation of candidate RBPs. Application to the HeLa RNA interactome yielded %CL values for 1,097 proteins. Remarkably, %CL values span over five orders of magnitude. For the HeLa RNA interactome, %CL values comprise a range from high efficiency, high specificity interactions, e.g., the Elav protein HuR and the Pumilio homolog Pum2, with %CL values of 45.9 and 24.2, respectively, to very low efficiency and specificity interactions e.g., the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and alpha-enolase, with %CL values of 0.0016, 0.006, and 0.008, respectively. We further extend the utility of %CL through prediction of protein domains and classes with known RNA-binding functions, thus establishing it as a useful metric for RNA interactome analysis. We anticipate that this approach will benefit efforts to establish functional RNA interactomes and support development of more predictive computational approaches for RNA binding protein identification.

中文翻译：

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蛋白质-RNA 紫外交联效率的高通量定量作为高可信度鉴定 RNA 结合蛋白的预测工具

紫外线交联已被证明是鉴定 RNA-蛋白质相互作用组的宝贵工具。然而，区分背景和真实 RNA-蛋白质相互作用的方法很少，这使得单独将 RNA 结合功能归因于 UV 交联具有挑战性。为了满足这一需求，我们之前报道了一种 RNA 结合蛋白 (RBP) 置信度评分指标 (RCS)，它结合了信噪比 (S:N) 和蛋白质丰度测定，以区分高置信度和低置信度候选 RBP。尽管 RCS 具有实用性，但我们寻求一种直接的指标来量化和比较评估蛋白质-RNA 相互作用。在这里，我们建议使用蛋白质特异性 UV 交联效率 (%CL)（代表与 RNA 交联的蛋白质的摩尔分数）来对候选 RBP 进行功能评估。应用 HeLa RNA 相互作用组得出 1,097 种蛋白质的 %CL 值。值得注意的是，%CL 值跨越了五个数量级。对于 HeLa RNA 相互作用组，%CL 值包括从高效率、高特异性相互作用（例如 Elav 蛋白 HuR 和 Pumilio 同源物 Pum2，%CL 值分别为 45.9 和 24.2）到极低效率和特异性相互作用的范围。例如，代谢酶甘油醛-3-磷酸脱氢酶、果糖二磷酸醛缩酶和α-烯醇酶，%CL 值分别为0.0016、0.006 和0.008。我们通过预测具有已知 RNA 结合功能的蛋白质结构域和类别，进一步扩展了 %CL 的实用性，从而将其确立为 RNA 相互作用组分析的有用指标。我们预计这种方法将有利于建立功能性 RNA 相互作用组的努力，并支持开发用于 RNA 结合蛋白识别的更具预测性的计算方法。