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

Leder V, Lummer M, Tegeler K, Humpert F, Lewinski M, Schüttpelz M, Staiger D (2014)
Biochemical and Biophysical Research Communications 453(1): 69-74.

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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.
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Leder V, Lummer M, Tegeler K, et al. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy. Biochemical and Biophysical Research Communications. 2014;453(1):69-74.
Leder, V., Lummer, M., Tegeler, K., Humpert, F., Lewinski, M., Schüttpelz, M., & Staiger, D. (2014). Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy. Biochemical and Biophysical Research Communications, 453(1), 69-74.
Leder, V., Lummer, M., Tegeler, K., Humpert, F., Lewinski, M., Schüttpelz, M., and Staiger, D. (2014). Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy. Biochemical and Biophysical Research Communications 453, 69-74.
Leder, V., et al., 2014. Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy. Biochemical and Biophysical Research Communications, 453(1), p 69-74.
V. Leder, et al., “Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy”, Biochemical and Biophysical Research Communications, vol. 453, 2014, pp. 69-74.
Leder, V., Lummer, M., Tegeler, K., Humpert, F., Lewinski, M., Schüttpelz, M., Staiger, D.: Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy. Biochemical and Biophysical Research Communications. 453, 69-74 (2014).
Leder, Verena, Lummer, Martina, Tegeler, Kathrin, Humpert, Fabian, Lewinski, Martin, Schüttpelz, Mark, and Staiger, Dorothee. “Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy”. Biochemical and Biophysical Research Communications 453.1 (2014): 69-74.
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Identification of the VERNALIZATION 4 gene reveals the origin of spring growth habit in ancient wheats from South Asia.
Kippes N, Debernardi JM, Vasquez-Gross HA, Akpinar BA, Budak H, Kato K, Chao S, Akhunov E, Dubcovsky J., Proc. Natl. Acad. Sci. U.S.A. 112(39), 2015
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