1. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy
- Author
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Martin Lewinski, Mark Schüttpelz, Fabian Humpert, Kathrin Tegeler, Verena Leder, Dorothee Staiger, and Martina Lummer
- Subjects
DNA, Plant ,RNA-binding protein ,Biophysics ,Arabidopsis ,Gene mutation ,Biology ,Genes, Plant ,Biochemistry ,Heterogeneous-Nuclear Ribonucleoproteins ,RNA-Protein Interaction ,RNA-protein-interaction ,Molecular Biology ,Conserved Sequence ,Binding Sites ,Base Sequence ,Arabidopsis Proteins ,Point mutation ,Alternative splicing ,Intron ,RNA-Binding Proteins ,Cell Biology ,Fluorescence correlation spectroscopy ,Molecular biology ,Introns ,Cell biology ,Alternative Splicing ,Spectrometry, Fluorescence ,RNA splicing ,Mutation ,Mutagenesis, Site-Directed ,Biologie ,Binding domain ,Protein Binding - Abstract
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 K-d 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. (C) 2014 Elsevier Inc. All rights reserved.
- Published
- 2014