Heterogeneous nuclear ribonucleoprotein U (hnRNP U) is a ubiquitously expressed protein that regulates chromatin architecture through its interactions with numerous DNA, protein, and RNA partners. The RNA-binding domain (RBD) of hnRNP U was previously mapped to an RGG/RG motif within its disordered C-terminal region, but little is understood about its binding mode and potential for selective RNA recognition. Analysis of publicly available hnRNP U enhanced UV cross-linking and immunoprecipitation (eCLIP) data identified high-confidence binding sites within human RNAs. We synthesized a set of diverse RNAs encompassing 11 of these identified cross-link sites for biochemical characterization using a combination of fluorescence anisotropy and electrophoretic mobility shift assays. These in vitro binding experiments with a rationally designed set of RNAs and hnRNP U domains revealed that the RGG/RG motif is a small part of a more expansive RBD that encompasses most of the disordered C-terminal region. This RBD contains a second, previously experimentally uncharacterized RGG/RG motif with RNA-binding properties comparable to those of the canonical RGG/RG motif. These RGG/RG motifs serve redundant functions, with neither serving as the primary RBD. While in isolation, each RGG/RG motif has modest affinity for RNA, together they significantly enhance the association of hnRNP U with RNA, enabling the binding of most of the designed RNA set with low to midnanomolar binding affinities. Identification and characterization of the complete hnRNP U RBD highlight the perils of a reductionist approach to defining biochemical activities in this system and pave the way for a detailed investigation of its RNA-binding specificity.

中文翻译：

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hnRNP U 的 RNA 结合域超出了 RGG/RG 基序

异质核核糖核蛋白 U (hnRNP U) 是一种普遍表达的蛋白质，通过与众多 DNA、蛋白质和 RNA 伴侣相互作用来调节染色质结构。hnRNP U 的 RNA 结合结构域 (RBD) 先前被映射到其无序 C 末端区域内的 RGG/RG 基序，但对其结合模式和选择性 RNA 识别的潜力知之甚少。对公开可用的 hnRNP U 增强型紫外交联和免疫沉淀 (eCLIP) 数据的分析确定了人类 RNA 内的高可信度结合位点。我们合成了一组不同的 RNA，其中包含 11 个已识别的交联位点，用于结合荧光各向异性和电泳迁移率变化测定进行生化表征。这些使用一组合理设计的 RNA 和 hnRNP U 结构域的体外结合实验表明，RGG/RG 基序是包含大部分无序 C 末端区域的更广泛的 RBD 的一小部分。该 RBD 包含第二个先前未通过实验表征的 RGG/RG 基序，其 RNA 结合特性与经典 RGG/RG 基序相当。这些 RGG/RG 基序具有冗余功能，两者都不充当主要 RBD。在分离时，每个 RGG/RG 基序对 RNA 具有适度的亲和力，它们一起显着增强 hnRNP U 与 RNA 的关联，使大多数设计的 RNA 组能够以低至中纳摩尔的结合亲和力结合。完整 hnRNP U RBD 的鉴定和表征强调了用还原论方法来定义该系统中的生化活性的危险，并为详细研究其 RNA 结合特异性铺平了道路。