Wang Lei, Shao Tian-Ci, Wang Chun-Ying, Li Jing-Jing, Jian Shao-Qin, Wang Duo, Cheng Bin-Feng
College of Life Science and Technology, Xinxiang Medical University, 453003 Xinxiang, Henan, China.
Front Biosci (Landmark Ed). 2023 Feb 8;28(2):24. doi: 10.31083/j.fbl2802024.
As a potent mediator of hypothermic neuroprotection, the cold-inducible protein RBM3 is characterized with one RNA-recognition motifs (RRM) and one arginine-glycine-rich (RGG) domain. It is known that these conserved domains are required for nuclear localization in some RNA-binding proteins. However, little is known about the actual role of RRM and RGG domains in subcellular localization of RBM3.
To clarify it, various mutants of human gene were constructed. Plasmids were transfected into cells and the localization of RBM3 protein and its varias mutants in cells and role in neuroprotection.
In human neuroblastoma SH-SY5Y cells, either a truncation of RRM domain (aa 1-86) or RGG domain (aa 87-157) led to an obvious cytoplasmic distribution, compared to a predominant nuclear localization of whole RBM3 protein (aa 1-157). In contrast, mutants in several potential phosphorylated sites of RBM3, including Ser102, Tyr129, Ser147, and Tyr155, did not alter the nuclear localization of RBM3. Similarly, mutants in two Di-RGG motif sites also did not affect the subcellular distribution of RBM3. Lastly, the role of Di-RGG motif in RGG domains was further investigated. The mutant of double arginines in either Di-RGG motif-1 (Arg87/90) or -2 (Arg99/105) exhibited a higher cytoplasmic localization, indicating that both Di-RGG motifs are required for nucleic localization of RBM3.
Our data suggest that RRM and RGG domains are both required for the nuclear localization of RBM3, with two Di-RGG domain being crucial for nucleocytoplasmic shuttling of RBM3.
作为低温神经保护的一种有效介质，冷诱导蛋白RBM3具有一个RNA识别基序（RRM）和一个富含精氨酸 - 甘氨酸的（RGG）结构域。已知这些保守结构域是某些RNA结合蛋白核定位所必需的。然而，关于RRM和RGG结构域在RBM3亚细胞定位中的实际作用知之甚少。
为阐明这一点，构建了人类基因的各种突变体。将质粒转染到细胞中，并研究RBM3蛋白及其各种突变体在细胞中的定位以及在神经保护中的作用。
在人神经母细胞瘤SH - SY5Y细胞中，与完整RBM3蛋白（1 - 157氨基酸）主要定位于细胞核相比，RRM结构域（1 - 86氨基酸）或RGG结构域（87 - 157氨基酸）的截短均导致明显的细胞质分布。相比之下，RBM3几个潜在磷酸化位点（包括Ser102、Tyr129、Ser147和Tyr155）的突变体并未改变RBM3的核定位。同样，两个双RGG基序位点的突变体也不影响RBM3的亚细胞分布。最后，进一步研究了RGG结构域中双RGG基序的作用。双RGG基序 - 1（Arg87 / 90）或 - 2（Arg99 / 105）中双精氨酸的突变体表现出更高的细胞质定位，表明两个双RGG基序都是RBM3核定位所必需的。
我们的数据表明，RRM和RGG结构域都是RBM3核定位所必需的，其中两个双RGG结构域对于RBM3的核质穿梭至关重要。
