The extended AT-hook is a novel RNA binding motif

The AT-hook has been defined as a DNA binding peptide motif that contains a glycine-arginine-proline (G-R-P) tripeptide core flanked by basic amino acids. Recent reports documented variations in the sequence of AT-hooks and revealed RNA binding activity of some canonical AT-hooks, suggesting a higher structural and functional variability of this protein domain than previously anticipated. Here we describe the discovery and characterization of the extended AT-hook peptide motif (eAT-hook), in which basic amino acids appear symmetrical mainly at a distance of 12–15 amino acids from the G-R-P core. We identified 80 human and 60 mouse eAT-hook proteins and biochemically characterized the eAT-hooks of Tip5/BAZ2A, PTOV1 and GPBP1. Microscale thermophoresis and electrophoretic mobility shift assays reveal the nucleic acid binding features of this peptide motif, and show that eAT-hooks bind RNA with one order of magnitude higher affinity than DNA. In addition, cellular localization studies suggest a role for the N-terminal eAT-hook of PTOV1 in nucleocytoplasmic shuttling. In summary, our findings classify the eAT-hook as a novel nucleic acid binding motif, which potentially mediates various RNA-dependent cellular processes.

AT-hook最初被定义为一类DNA结合肽基序，其核心为甘氨酸-精氨酸-脯氨酸（G-R-P）三肽结构，侧翼带有碱性氨基酸残基。近期研究报道了AT-hook序列的变异，并揭示了部分经典AT-hook的RNA结合活性，提示该蛋白质结构域的结构与功能多样性远超此前预期。本研究报道了延伸型AT-hook肽基序（eAT-hook）的发现与表征：该基序中的碱性氨基酸主要以对称方式分布在距G-R-P核心12~15个氨基酸的位置。我们共鉴定出80个人类和60个小鼠的eAT-hook蛋白，并对Tip5/BAZ2A、PTOV1及GPBP1的eAT-hook进行了生化表征。通过微量热泳动与电泳迁移率变动分析，我们揭示了该肽基序的核酸结合特性，并证实eAT-hook与RNA的结合亲和力较DNA高出一个数量级。此外，细胞定位研究提示，PTOV1的N端eAT-hook参与核质穿梭过程。总而言之，本研究将eAT-hook归类为一类新型核酸结合基序，其可能介导多种依赖RNA的细胞过程。