G-Quadruplex structural motifs modulate protein-RNA interactions within the transcriptome

RNA secondary structures, including G-quadruplexes (G4s), have emerged as vital players in protein–RNA interactions. The RNA-binding protein Fused in Sarcoma (FUS), which is strongly implicated in both neurodegenerative disease and cancer, is known to interact with RNA molecules through a variety of GU-rich sequences. However, a definitive consensus motif for FUS–RNA recognition and binding has not yet been determined. Here, we hypothesize that G4 structures, which are inherently G-rich, may play a key role in FUS binding. We examine the role of G4s in FUS–RNA binding by developing an RNA immunoprecipitation sequencing (RIP-seq) protocol under G4-stabilising and non-stabilising conditions. We find that G4s regulate the binding of FUS to target RNAs, providing new information on protein–RNA binding motifs, while reinforcing the importance of RNA secondary structures as pivotal regulators of protein interactions. These insights advance our understanding of FUS-RNA binding dynamics and future potential for identifying new therapeutic targets for neurodegenerative disease and other FUS-related pathologies. Overall design: RNA G4 secondary structures were annealed in RIP buffer containing 150 mM of K+ or Li+, then bound to recombinant C-terminal FUS protein. FUS-bound RNA was purified and next-generation sequencing performed to identify G4-dependent FUS-RNA binding. Input samples were subject to all steps as FUS-RNA samples except for immunoprecipitation and subsequent washes. Each condition was performed in triplicate.

RNA二级结构，包括G-四链体（G-quadruplexes，G4s），现已成为蛋白质-RNA相互作用中的关键调控因子。RNA结合蛋白肉瘤融合蛋白（Fused in Sarcoma，FUS）与神经退行性疾病及癌症均密切相关，已知可通过多种富含GU的序列与RNA分子结合。然而，目前尚未明确FUS识别并结合RNA的确定共识基序。本研究推测，天然富含G的G4结构可能在FUS与RNA的结合过程中发挥关键作用。为此，我们通过在G4稳定化与非稳定化条件下构建RNA免疫共沉淀测序（RNA immunoprecipitation sequencing，RIP-seq）实验方案，探究G4在FUS-RNA结合过程中的调控作用。研究发现，G4可调控FUS与靶标RNA的结合，这为蛋白质-RNA结合基序研究提供了新的认知，同时进一步证实RNA二级结构作为蛋白质相互作用关键调控因子的重要性。这些研究发现加深了我们对FUS-RNA结合动态机制的理解，也为未来识别神经退行性疾病及其他FUS相关疾病的新型治疗靶点提供了潜在方向。实验整体设计：将RNA G4二级结构在含150 mM钾离子（K+）或锂离子（Li+）的RIP缓冲液中退火处理，随后与重组C端FUS蛋白结合。纯化结合FUS的RNA并进行下一代测序（next-generation sequencing），以鉴定依赖G4结构的FUS-RNA结合事件。输入样本除未进行免疫共沉淀及后续洗涤步骤外，其余操作与FUS-RNA样本完全一致。所有实验条件均设置三次生物学重复。