Gemin5 is a multifunctional RNA binding protein (RBP) organized in domains with a distinctive structural organization. The protein is a hub for several protein networks performing diverse RNA-dependent functions including regulation of translation, and recognition of small nuclear RNAs (snRNAs). Here we sought to identify the presence of phosphoresidues on the C-terminal half of Gemin5, a region of the protein that harbors a tetratricopeptide repeat (TPR)-like dimerization domain and a non-canonical RNA binding site (RBS1). We identified two phosphoresidues in the purified protein: P-T897 in the dimerization domain and P-T1355 in RBS1. Replacing T897 and T1355 with alanine led to decreased translation, and mass spectrometry analysis revealed that mutation T897A strongly abrogates the association with cellular proteins related to the regulation of translation. In contrast, the phosphomimetic substitutions to glutamate partially rescued the translation regulatory activity. The structural analysis of the TPR dimerization domain indicates that local rearrangements caused by phosphorylation of T897 affect the conformation of the flexible loop 2-3, and propagate across the dimerization interface, impacting the position of the C-terminal helices and the loop 12-13 shown to be mutated in patients with neurological disorders. Computational analysis of the potential relationship between post-translation modifications and currently known pathogenic variants indicates a lack of overlapping of the affected residues within the functional domains of the protein and provides molecular insights for the implication of the phosphorylated residues in translation regulation.

中文翻译：

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Gemin5 二聚化结构域中 T897 的磷酸化可调节蛋白质相互作用和翻译调节。

Gemin5 是一种多功能 RNA 结合蛋白 (RBP)，在具有独特结构组织的域中组织。该蛋白质是多个蛋白质网络的枢纽，执行多种 RNA 依赖性功能，包括翻译调节和小核 RNA (snRNA) 识别。在这里，我们试图确定 Gemin5 的 C 末端一半上是否存在磷酸残基，该蛋白质区域包含四肽重复序列 (TPR) 样二聚化结构域和非规范 RNA 结合位点 (RBS1)。我们在纯化的蛋白质中鉴定了两个磷酸残基：二聚化结构域中的 P-T897 和 RBS1 中的 P-T1355。用丙氨酸替换 T897 和 T1355 导致翻译减少，和质谱分析表明，突变 T897A 强烈废除了与翻译调节相关的细胞蛋白的关联。相反，对谷氨酸的拟磷酸化取代部分挽救了翻译调节活性。TPR 二聚化结构域的结构分析表明，由 T897 磷酸化引起的局部重排影响柔性环 2-3 的构象，并传播穿过二聚化界面，影响 C 末端螺旋和环 12-13 的位置显示在患有神经系统疾病的患者中发生突变。