Study of Stress Granule Core Protein AtUBP1b Phosphorylation In Vitro
Nizkorodova A.S. Kislitsin V.Y. Zhigailov A.V. Kulyyassov A.T. Nadirova L.M. Stanbekova G.E. Iskakov B.K.
October 2025Multidisciplinary Digital Publishing Institute (MDPI)
Plants
2025#14Issue 20
Stress granules (SGs) are dynamic membrane-less structures assembled in response to stress. The formation of stress granules in plants is poorly understood, especially the mechanism of mRNA recruitment. The problem of the specificity of mRNA interaction with stress granule proteins is unexplored. Oligouridylate binding protein 1B (UBP1B) is considered as the core element of plant SGs. In this study, we expressed the AtUBP1b protein from Arabidopsis thaliana in E. coli cells. Mass spectroscopic analysis showed that the AtUBP1b protein expressed in E. coli cells is phosphorylated at serine, threonine, and tyrosine residues. We also performed a de novo phosphorylation reaction in wheat germ extracts with the addition of radioactively labeled phosphorus and showed AtUBP1b phosphorylation in plant extracts. We hypothesized that phosphorylation or dephosphorylation of AtUBP1b in plant cells is a signal for protein binding to RNA. The purified protein was tested for its ability to bind to mRNA in vitro. In gel-shifting assays we demonstrated that AtUBP1b protein binds specifically to 5′-untranslated regions (5′UTR) of mRNA. When AtUBP1b was added to a cell-free wheat germ translation system, it exerted different effects on protein synthesis. We showed that AtUBP1b had a significant inhibitory effect on the expression of mRNAs containing 5′UTRs that were shown to bind to the protein in the gel-shifting reaction.
AtUBP1b , gel-shifting , phosphorylation , RNA binding protein , stress granules
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M. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty, 050012, Kazakhstan
National Center of Biotechnology, Astana, 010000, Kazakhstan
M. Aitkhozhin Institute of Molecular Biology and Biochemistry
National Center of Biotechnology
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