Guzmán Bryan B, Goda Grant A, Jimenez Alli, Martyr Justin G, Hu Yue, Cavazos Francisco F, Aleman Maria M, Dominguez Daniel
Department of Pharmacology, University of North Carolina, Chapel Hill, NC.
Department of Chemistry, University of North Carolina, Chapel Hill, NC.
bioRxiv. 2025 May 1:2025.05.01.651718. doi: 10.1101/2025.05.01.651718.
RNA binding proteins (RBPs) interact with and tightly regulate the fate of messenger RNAs but how RNA targets are recognized remains a challenging question. RBPs often contain multiple domains known to directly bind RNA, such as RNA recognition motifs (RRMs), as well as domains whose RNA binding capacity remains incompletely understood, low complexity domains (LCDs). Here, we dissect HNRNPR, an RBP with three RRMs and an arginine-glycine rich (RG-rich) LCD. We apply unbiased high-throughput biochemical approaches and identify critical RNA binding domains that confer specificity. We show that not all RRMs contribute equally to binding and find that RRM3, along with a downstream C-terminal charged region, are required for RNA binding. We find that HNRNPR also binds RNA G-quadruplexes (rG4s) and map multiple rG4 binding sites including RRM3 with the C-terminal charged region and RG-rich regions within the LCD. We dissect rG4 specificity for the full length HNRNPR and LCD using a newly created RNA pool focused on rG4s and reveal that binding is dependent on RNA folding and find specific rG4 features that enhance HNRNPR-rG4 interactions. Our work highlights the complexity of RBP-RNA interactions and motivates the study of disordered regions as RNA binding domains.
RNA结合蛋白（RBPs）与信使RNA相互作用并严格调控其命运，但RNA靶点是如何被识别的仍是一个具有挑战性的问题。RBPs通常包含多个已知可直接结合RNA的结构域，如RNA识别基序（RRMs），以及RNA结合能力尚不完全清楚的结构域，即低复杂性结构域（LCDs）。在这里，我们剖析了HNRNPR，一种具有三个RRMs和一个富含精氨酸 - 甘氨酸（RG-rich）的LCD的RBP。我们应用无偏差的高通量生化方法，鉴定出赋予特异性的关键RNA结合结构域。我们表明并非所有RRMs对结合的贡献都相同，并发现RRM3以及下游的C末端带电荷区域是RNA结合所必需的。我们发现HNRNPR还结合RNA G-四链体（rG4s），并绘制了多个rG4结合位点，包括带有C末端带电荷区域的RRM3以及LCD内的富含RG区域。我们使用新创建的聚焦于rG4s的RNA库剖析了全长HNRNPR和LCD对rG4的特异性，揭示结合依赖于RNA折叠，并发现了增强HNRNPR-rG4相互作用的特定rG4特征。我们的工作突出了RBP-RNA相互作用的复杂性，并激发了对作为RNA结合结构域的无序区域的研究。
