RNA剪接
生物
绑定域
结合位点
突变体
点突变
荧光相关光谱
拟南芥
寡核苷酸
遗传学
生物化学
内含子
RNA结合蛋白
拟南芥
核糖核酸
分子生物学
基因
化学
有机化学
分子
作者
Verena Leder,Martina Lummer,Kathrin Tegeler,Fabian Humpert,Martin Lewinski,Mark Schüttpelz,Dorothee Staiger
标识
DOI:10.1016/j.bbrc.2014.09.056
摘要
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 R49 that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.
科研通智能强力驱动
Strongly Powered by AbleSci AI