Vidal Ceballos Alfredo, Geissmann Anna, Favaro Denize C, Deshpande Priyasha, Elbaum-Garfinkle Shana
Structural Biology Initiative, Advanced Science Research Center, CUNY, New York, NY, USA.
Ph.D. Program in Chemistry, Graduate Center of the City University of New York, New York, NY, USA.
Sci Rep. 2025 Mar 18;15(1):9295. doi: 10.1038/s41598-025-88499-y.
RNA binding proteins (RBPs) are enriched in phase separated biomolecular assemblies across cell types. These RBPs often harbor arginine-glycine rich RGG motifs, which can drive phase separation, and can preferentially interact with RNA G-quadruplex (G4) structures, particularly in the neuron. Increasing evidence underscores the important role that RNA sequence and structure play in contributing to the form and function of protein condensates, however, less is known about the role of G4 RNAs and their interaction with RGG domains specifically. In this study we focused on the model protein, Fragile X mental retardation protein (FMRP), to investigate how G4-containing RNA sequences impact the phase behavior and material properties of condensates. FMRP is implicated in the development of Fragile X Syndrome, and is enriched in neuronal granules where it is thought to aid in mRNA trafficking and translational control. Here, we examined RNA sequences with increasing G content and G4 propensity in complex with the RGG-containing low complexity region (LCR) of FMRP. We found, that while increasing G content triggers aggregation of poly-arginine, all RNA sequences supported phase separation into liquid droplets with FMRP-LCR. Combining microrheology, and fluorescence recovery after photobleaching, we measured a moderate increase in viscosity and decrease in dynamics for increasing G-content, and detected no measurable increase in elasticity as a function of G4 structure. Additionally, we found that while methylation of FMRP decreased RNA binding affinity, this modification did not impact condensate material properties suggesting that RNA sequence/structure can play a greater role than binding affinity in determining the emergent properties of condensates. Together, this work lends much needed insight into the ways in which G-rich RNA sequences tune the assembly, dynamics and material properties of protein/RNA condensates and/or granules.
RNA结合蛋白（RBPs）在跨细胞类型的相分离生物分子聚集体中富集。这些RBPs通常含有富含精氨酸-甘氨酸的RGG基序，其可驱动相分离，并可优先与RNA G-四链体（G4）结构相互作用，尤其是在神经元中。越来越多的证据强调了RNA序列和结构在促成蛋白质凝聚物的形式和功能方面所起的重要作用，然而，关于G4 RNA的作用及其与RGG结构域的特异性相互作用了解较少。在本研究中，我们聚焦于模型蛋白脆性X智力低下蛋白（FMRP），以研究含G4的RNA序列如何影响凝聚物的相行为和材料特性。FMRP与脆性X综合征的发生有关，并且在神经元颗粒中富集，据认为它在mRNA运输和翻译控制中发挥作用。在此，我们研究了与FMRP含RGG的低复杂性区域（LCR）复合时，G含量和G4倾向增加的RNA序列。我们发现，虽然增加G含量会触发聚精氨酸的聚集，但所有RNA序列都支持与FMRP-LCR相分离成液滴。结合微观流变学和光漂白后的荧光恢复，我们测量了随着G含量增加粘度适度增加和动力学降低，并且未检测到作为G4结构函数的弹性有可测量的增加。此外，我们发现虽然FMRP的甲基化降低了RNA结合亲和力，但这种修饰并未影响凝聚物材料特性，这表明RNA序列/结构在决定凝聚物的涌现特性方面比结合亲和力可能发挥更大的作用。总之，这项工作为富含G的RNA序列调节蛋白质/RNA凝聚物和/或颗粒的组装、动力学和材料特性的方式提供了急需的见解。
