Leder Verena, Lummer Martina, Tegeler Kathrin, Humpert Fabian, Lewinski Martin, Schüttpelz Mark, Staiger Dorothee
Molecular Cell Physiology, Faculty of Biology, Bielefeld University, Germany; Biomolecular Photonics, Faculty of Physics, Bielefeld University, Germany.
Molecular Cell Physiology, Faculty of Biology, Bielefeld University, Germany.
Biochem Biophys Res Commun. 2014 Oct 10;453(1):69-74. doi: 10.1016/j.bbrc.2014.09.056. Epub 2014 Sep 22.
Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased Kd value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R(49) that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.
拟南芥富含甘氨酸的RNA结合蛋白7（AtGRP7）是负反馈回路的一部分，通过该回路它调节其前体mRNA的可变剪接和稳态丰度。在这里，我们使用荧光相关光谱法，通过荧光标记的合成寡核苷酸研究AtGRP7与其内含子结合的要求。通过系统地引入点突变，我们鉴定出三个核苷酸，当它们发生突变时会导致解离常数（Kd）值增加，因此对AtGRP7结合至关重要。所有三个残基的同时突变会消除结合。旁系同源物AtGRP8与重叠基序结合，但具有不同的序列偏好，这与该蛋白对的重叠但不相同的功能一致。富含甘氨酸结构域的截短降低了AtGRP7的结合亲和力，首次表明植物hnRNP样蛋白的富含甘氨酸区域有助于结合。对病原体防御和剪接中AtGRP7功能至关重要的保守R（49）突变会消除结合。
