Institute of Biochemistry, Charite - University Medicine Berlin, Corporate member of Free University Berlin, Humboldt University Berlin and Berlin Institute of Health, Charitéplatz 1, D-10117 Berlin, Germany.
Institute of Biochemistry, Charite - University Medicine Berlin, Corporate member of Free University Berlin, Humboldt University Berlin and Berlin Institute of Health, Charitéplatz 1, D-10117 Berlin, Germany.
Biochim Biophys Acta Gen Subj. 2020 Nov;1864(11):129678. doi: 10.1016/j.bbagen.2020.129678. Epub 2020 Jul 6.
The guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the hnRNP H/F family, which has been implicated in erythropoiesis, regulation of the redox homeostasis, embryonic brain development, mitochondrial function and cellular senescence. The molecular basis for GRSF1-RNA interaction has extensively been studied in the past but for the time being GRSF1 binding proteins have not been identified.
To search for GRSF1 binding proteins we first employed the yeast two-hybrid system and screened a cDNA library of human fetal brain for potential GRSF1 binding proteins. Subsequently, we explored the protein-protein-interaction of the recombiant proteins, carried out immunoprecipitation experiments to confirm the interaction of the native proteins in living cells and performed truncation studies to identify the protein-binding motif of GRSF1.
Using the yeast two-hybrid system we identified the COMM-domain containing protein 1 (COMMD1) as specific GRSF1 binding protein and in vitro truncation studies suggested that COMMD1 interacts with the alanine-rich domain of GRSF1. Co-immunoprecipitation strategies indicated that COMMD1-GRSF1 interaction was RNA independent and also occurred in living cells expressing the two native proteins.
In mammalian cells the COMM-domain containing protein 1 (COMMD1) specifically interacts with the Ala-rich domain of GRSF1 in an RNA-independent manner.
This is the first report describing a specific GRSF1 binding protein.
富含鸟嘌呤的 RNA 序列结合因子 1（GRSF1）是 hnRNP H/F 家族的 RNA 结合蛋白，它与红细胞生成、氧化还原稳态调节、胚胎大脑发育、线粒体功能和细胞衰老有关。过去已经广泛研究了 GRSF1-RNA 相互作用的分子基础，但目前尚未鉴定出 GRSF1 结合蛋白。
为了寻找 GRSF1 结合蛋白，我们首先使用酵母双杂交系统筛选了人胎脑 cDNA 文库中的潜在 GRSF1 结合蛋白。随后，我们探索了重组蛋白的蛋白质-蛋白质相互作用，进行免疫沉淀实验以确认活细胞中天然蛋白的相互作用，并进行截短研究以鉴定 GRSF1 的蛋白结合基序。
使用酵母双杂交系统，我们鉴定了 COMM 结构域包含蛋白 1（COMMD1）为特定的 GRSF1 结合蛋白，体外截短研究表明 COMMD1 与 GRSF1 的富含丙氨酸结构域相互作用。共免疫沉淀策略表明 COMMD1-GRSF1 相互作用不依赖于 RNA，并且在表达两种天然蛋白的活细胞中也发生。
在哺乳动物细胞中，COMM 结构域包含蛋白 1（COMMD1）以不依赖于 RNA 的方式特异性地与 GRSF1 的 Ala 丰富结构域相互作用。
这是首次描述特定的 GRSF1 结合蛋白的报告。
