title: Interaction with Cu²⁺ disrupts the RNA binding affinities of RNA recognition motif containing protein.
abstract: The glycine-rich proteins (GRP) containing RNA recognition motifs (RRM) are involved in the regulation of transcriptional and/or post-transcriptional events. Previous studies have established that GRP162 plays an important role in the restoration of fertility in Honglian cytoplasmic male sterile (HL-CMS) rice. In this study, the ion binding properties of rGRP162 were tested by isothermal titration calorimetry (ITC) and electrophoretic mobility shift assay (EMSA) was performed to test the interaction. Circular dichroism (CD) was carried out to detect the alteration of secondary structure in the presence and absence of Cu(2+). Furthermore, two RRM containing proteins, AtRBP45A and AtRBP47A, were expressed to validate the interaction. Results showed Cu(2+) and Fe(3+) bound GRP162, whereas Ca(2+), Mn(2+), Mg(2+) and K(+) did not. EMSA confirmed that interaction with Cu(2+) interrupted the biological activity of GRP162 by disrupting the secondary structure of the protein based on the results of CD. Moreover, the RNA binding activities of rAtRBP45A and rAtRBP47A were also impaired in the presence of Cu(2+). Data suggest that Cu(2+) in excess may disrupt RNA-binding proteins containing RRM that are essential for post-transcriptional regulation and may impair the development of plants or animals.
authors.affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China; Engineering Research Center for Plant Biotechnology and Germplasm, Utilization, Ministry of Education, Wuhan University, Wuhan, China.
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China; Engineering Research Center for Plant Biotechnology and Germplasm, Utilization, Ministry of Education, Wuhan University, Wuhan, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China; Engineering Research Center for Plant Biotechnology and Germplasm, Utilization, Ministry of Education, Wuhan University, Wuhan, China.
authors.forename: Linlin
authors.initials: L
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China; Engineering Research Center for Plant Biotechnology and Germplasm, Utilization, Ministry of Education, Wuhan University, Wuhan, China.
authors.forename: Haijun
authors.initials: H
authors.lastname: Xiao
authors.affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China; Engineering Research Center for Plant Biotechnology and Germplasm, Utilization, Ministry of Education, Wuhan University, Wuhan, China.
authors.forename: Guoxin
authors.initials: G
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China; Engineering Research Center for Plant Biotechnology and Germplasm, Utilization, Ministry of Education, Wuhan University, Wuhan, China.
authors.forename: Wenchao
authors.initials: W
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China; Engineering Research Center for Plant Biotechnology and Germplasm, Utilization, Ministry of Education, Wuhan University, Wuhan, China.
authors.forename: Renshan
authors.initials: R
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China; Engineering Research Center for Plant Biotechnology and Germplasm, Utilization, Ministry of Education, Wuhan University, Wuhan, China. Electronic address: junhu@whu.edu.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China; Engineering Research Center for Plant Biotechnology and Germplasm, Utilization, Ministry of Education, Wuhan University, Wuhan, China. Electronic address: zhuyg@public.wh.hb.cn.
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Zhu
mesh_terms: D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002151:Calorimetry; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D012275:Oryza / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D010940:Plant Proteins; C518408:RBP45a protein, Arabidopsis; C588091:RBP47A protein, Arabidopsis; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA; D003300:Copper
keywords: Copper; Glycine-rich protein; RNA recognition motif (RRM); RNA-binding affinity