title: Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy.
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 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.
authors.affiliation: Molecular Cell Physiology, Faculty of Biology, Bielefeld University, Germany; Biomolecular Photonics, Faculty of Physics, Bielefeld University, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Leder
authors.affiliation: Molecular Cell Physiology, Faculty of Biology, Bielefeld University, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Lummer
authors.affiliation: Molecular Cell Physiology, Faculty of Biology, Bielefeld University, Germany; Biomolecular Photonics, Faculty of Physics, Bielefeld University, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Tegeler
authors.affiliation: Biomolecular Photonics, Faculty of Physics, Bielefeld University, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Humpert
authors.affiliation: Molecular Cell Physiology, Faculty of Biology, Bielefeld University, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Lewinski
authors.affiliation: Biomolecular Photonics, Faculty of Physics, Bielefeld University, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Schüttpelz
authors.affiliation: Molecular Cell Physiology, Faculty of Biology, Bielefeld University, Germany. Electronic address: dorothee.staiger@uni-bielefeld.de.
authors.forename: Dorothee
authors.initials: D
authors.lastname: Staiger
mesh_terms: D017398:Alternative Splicing; D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D018744:DNA, Plant / Q000235:genetics; D017343:Genes, Plant; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007438:Introns; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D029681:Arabidopsis Proteins; D018744:DNA, Plant; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D016601:RNA-Binding Proteins; C118496:GRP7 protein, Arabidopsis
keywords: Fluorescence correlation spectroscopy; RNA-binding protein; RNA–protein-interaction