Abstract：
The abundant nuclear protein hnRNP U interacts with a broad array of RNAs along with DNA and protein to regulate nuclear chromatin architecture. The RNA-binding activity is achieved via a disordered ∼100 residue C-terminal RNA-binding domain (RBD) containing two distinct RGG/RG motifs. Although the RNA-binding capabilities of RGG/RG motifs have been widely reported, less is known about hnRNP U\'s RNA-binding selectivity. Furthermore, while it is well established that hnRNP U binds numerous nuclear RNAs, it remains unknown whether it selectively recognizes sequence or structural motifs in target RNAs. To address this question, we performed equilibrium binding assays using fluorescence anisotropy (FA) and electrophoretic mobility shift assays (EMSAs) to quantitatively assess the ability of human hnRNP U RBD to interact with segments of cellular RNAs identified from eCLIP data. These RNAs often, but not exclusively, contain poly-uridine or 5\'-AGGGAG sequence motifs. Detailed binding analysis of several target RNAs reveal that the hnRNP U RBD binds RNA in a promiscuous manner with high affinity for a broad range of structured RNAs, but with little preference for any distinct sequence motif. In contrast, the isolated RGG/RG of hnRNP U motif exhibits a strong preference for G-quadruplexes, similar to that observed for other RGG motif bearing peptides. These data reveal that the hnRNP U RBD attenuates the RNA binding selectivity of its core RGG motifs to achieve an extensive RNA interactome. We propose that a critical role of RGG/RG motifs in RNA biology is to alter binding affinity or selectivity of adjacent RNA-binding domains.
摘要：
丰富的核蛋白hnRNPU与广泛的RNA以及DNA和蛋白质相互作用以调节核染色质结构。RNA结合活性是通过含有两个不同的RGG/RG基序的无序~100残基C端RNA结合域（RBD）实现的。尽管RGG/RG基序的RNA结合能力已被广泛报道，对hnRNPU的RNA结合选择性知之甚少。此外，虽然已经确定hnRNPU结合许多核RNA，尚不清楚它是否选择性识别靶RNA中的序列或结构基序。为了解决这个问题，我们使用荧光各向异性（FA）和电泳迁移率变化测定（EMSAs）进行了平衡结合测定，以定量评估人类hnRNPURBD与从eCLIP数据中鉴定的细胞RNA片段相互作用的能力。这些RNA经常，但不限于此,含有聚尿苷或5'-AGGGAG序列基序。对几种靶RNA的详细结合分析表明，hnRNPURBD以混杂的方式结合RNA，对广泛的结构化RNA具有高亲和力，但很少偏爱任何不同的序列基序。相比之下,hnRNPU基序的分离的RGG/RG表现出对G-四链体的强烈偏好，与其他带有RGG基序的肽所观察到的相似。这些数据表明，hnRNPURBD削弱了其核心RGG基序的RNA结合选择性，从而实现了广泛的RNA相互作用。我们建议RGG/RG基序在RNA生物学中的关键作用是改变相邻RNA结合结构域的结合亲和力或选择性。
