Institute of Neuroscience and the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou 510260, China.
Institute of Neuroscience and the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou 510260, China; State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing 100875, China.
Neuroscience. 2019 Apr 15;404:282-296. doi: 10.1016/j.neuroscience.2019.01.028. Epub 2019 Feb 10.
Fragile X mental retardation protein (FMRP), a key determinant of normal brain development and neuronal plasticity, plays critical roles in nucleocytoplasmic shuttling of mRNAs. However, the factors involved in FMRP nuclear localization remain to be determined. Using cross-species sequence comparison, we show that an aspartate in position 132 (D132), located within the conserved nuclear localization signal (NLS) of FMRP, appears in human and other mammals, while glutamate 132 (E132) appears in rodents and birds. Human FMRP-D132E alters the secondary structure of the protein and reduces its nuclear localization, while the reciprocal substitution in mouse FMRP-E132D promotes its nuclear localization. Human FMRP could interact with poly(A)-binding protein 1 (PABP1) which is impeded by the D132E mutation. Reversely, mouse FMRP could not interact with PABP1, but the E132D mutation leads to the FMRP-PABP1 interaction. We further show that overexpression of human FMRP-D132E mutant promotes the formation of cytoplasmic aggregates of PABP1 in human cells, but not of mouse FMRP-E132D in mouse cells. PABP1 knockdown reduces the nuclear localization of human FMRP, but not mouse FMRP. Furthermore, RNase A treatment decreases the PABP1 levels in the anti-V5-immunoprecipitates using the V5-hFMRP-transfected cells, suggesting an interaction between human FMRP and PABP1 in an RNA-dependent fashion. Thus, our data suggest that the FMRP protein with the human-used D132 accommodates a novel protein-RNA-protein interaction which may implicate a connection between FMRP residue transition and neural evolution.
脆性 X 智力低下蛋白（FMRP）是正常大脑发育和神经元可塑性的关键决定因素，在 mRNA 的核质穿梭中发挥着关键作用。然而，FMRP 核定位所涉及的因素仍有待确定。通过跨物种序列比较，我们发现位于 FMRP 保守核定位信号（NLS）内的 132 位天冬氨酸（D132）出现在人类和其他哺乳动物中，而谷氨酸 132（E132）出现在啮齿动物和鸟类中。人类 FMRP-D132E 改变了蛋白质的二级结构并减少了其核定位，而在小鼠 FMRP-E132D 中的相反取代则促进了其核定位。人类 FMRP 可以与多聚（A）结合蛋白 1（PABP1）相互作用，而 D132E 突变会阻碍这种相互作用。相反，小鼠 FMRP 不能与 PABP1 相互作用，但 E132D 突变会导致 FMRP-PABP1 相互作用。我们进一步表明，过表达人类 FMRP-D132E 突变体可促进 PABP1 在人类细胞中的细胞质聚集体形成，但不会促进小鼠 FMRP-E132D 在小鼠细胞中的聚集体形成。PABP1 的敲低减少了人类 FMRP 的核定位，但不会减少小鼠 FMRP 的核定位。此外，用转染了 V5-hFMRP 的细胞进行的 RNase A 处理降低了抗 V5-免疫沉淀中的 PABP1 水平，表明人类 FMRP 与 PABP1 之间存在 RNA 依赖性相互作用。因此，我们的数据表明，具有人类使用的 D132 的 FMRP 蛋白容纳了一种新的蛋白质-RNA-蛋白质相互作用，这可能暗示了 FMRP 残基转变与神经进化之间的联系。
