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Title: Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

Highlights: • We use FCS to investigate binding site requirements for the hnRNP-like protein AtGRP7. • We identify three nucleotides critical for AtGRP7 binding to its own intron. • Mutation of the conserved R{sup 49} abolishes binding altogether. • The paralogue AtGRP8 binds to an overlapping motif with different sequence requirement. • The glycine-rich stretch of a plant hnRNP-like protein contributes to 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{sub 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.more » Mutation of the conserved R{sup 49} that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.« less
Authors:
 [1] ;  [2] ;  [1] ;  [1] ;  [2] ;  [3] ;  [1] ;  [3] ;  [1]
  1. Molecular Cell Physiology, Faculty of Biology, Bielefeld University (Germany)
  2. (Germany)
  3. Biomolecular Photonics, Faculty of Physics, Bielefeld University (Germany)
Publication Date:
OSTI Identifier:
22416782
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 453; Journal Issue: 1; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AFFINITY; ARABIDOPSIS; CORRELATIONS; FEEDBACK; FLUORESCENCE SPECTROSCOPY; GENE MUTATIONS; GLYCINE; MESSENGER-RNA; NUCLEOTIDES; OLIGONUCLEOTIDES; PATHOGENS; PROTEINS; RESIDUES; SPLICING; STEADY-STATE CONDITIONS