Project AP19676956 “Investigation of the formation of plant stress granules by RNA-binding proteins AtURB1b and AtRBP47a from Arabidopsis thaliana”.
The Project description
One of the fundamental problems of modern molecular biology is the cellular response to stress. The stability and viability of the entire organism as a whole depends on the cell’s response to stress at the molecular level. The response to stress in all eukaryotic cells is the formation of extramembrane stress granules (SGs) in the cytoplasm, consisting of RNA-protein complexes. Mandatory components of these granules are RNA-binding proteins belonging to the TIA-1/TIAR family of animal cells. Their homologues in plants are proteins of the UBP1 and RBP45/47 families. In plants, the mechanisms of formation of SGs and their RNA-binding proteins have been studied to a lesser extent than in animals and yeast. This project will investigate the RNA-binding proteins AtUBP1b and AtRBP47a from Arabidopsis thaliana, their role in the formation of stress granules and in the regulation of general protein synthesis under various stresses.
The theory of the process of formation of SGs currently continues to be developed, since there are a number of fundamental questions to which there are no answers yet. The study of SGs and the proteins that form them is also important from an applied point of view. Thus, in Arabidopsis plants it was shown that an increase in the expression of the AtUBP1b protein leads to increased plant resistance to heat stress, and the AtUBP1c protein to hypoxia conditions. In Nicotiana tabacum plants, it was shown that with increased expression of the NtRBP45 protein, the effect of RNA silencing against the tobacco mosaic virus that infected the plants increased, that is, plant immunity was enhanced.
In the course of the proposed study, data will be obtained that will clarify the role of the AtUBP1b and AtRBP47a proteins in the plant cell. Understanding the functions of these proteins will allow new approaches to improve plant resistance to stress.
The idea of the project is the translation of AtUBP1b and AtRBP47a proteins in three expression systems: bacterial (E. coli), transient plant (N. benthamiana) and constitutive plant (A. thaliana). Proteins produced in E. coli cells will be used for a series of in vitro experiments. Protein complexes isolated from tobacco will be examined for the composition of RNA and proteins (and their possible modifications). In Arabidopsis, the physiological response to various types of stress will be studied against the background of enhanced expression of AtUBP1b and AtRBP47a.
The Project goals
To establish the functional role of the RNA-binding proteins AtUBP1b and AtRBP47a from A. thaliana in the reversible formation of stress granules in plant cells.
The Project objectives
- Cloning the cDNA genes of two Arabidopsis proteins, AtUbp1b and AtRbp47a, into a bacterial expression vector.
- Express cDNA genes in Escherichia coli and purify target proteins using IMAC chromatography (Immobilized Metal ion Affinity Chromatography)
- Using the “gel-shifting” approach, study in vitro the interaction of isolated target proteins with reporter mRNAs having various 5’- and 3’-NTP variants.
- To study the influence of target proteins on the process of in vitro translation of reporter mRNAs with various 5′- and 3′-NTP variants in a cell-free system under normal conditions and under a number of stresses.
- Clone the AtUbp1b and AtRbp47a sequences into a vector for transient transformation of Nicotiana benthamiana plants and into a vector for constitutive transformation of Arabidopsis thaliana.
- In Arabidopsis F1 plants transformed by the “floral dip” method, evaluate the level of expression of transgenic inserts and subject these plants to a series of stresses, during which the physiological parameters of the plants will be assessed.
- Subject transformed benthamiana plants to various types of stress, isolate target proteins (IMAC) and evaluate their covalent modification (phosphorylation) using two-dimensional electrophoresis relative to intact plants.
- To study the distribution of target proteins in subcellular fractions in transformed tobacco plants before and after stress treatment. Using sedimentation analysis in a gradient of sucrose concentrations, we studied the possibility of the inclusion of AtUBP1b and AtRBP47a in the composition of polysome fractions, 80S, 60S and 40S in transformed tobacco plants before and after stress.
- From the subcellular fractions in which the target proteins are found, use immunoprecipitation to isolate the AtUBP1b and AtRBP47a protein complexes; elute bound RNA from these complexes and analyze their composition (RT-PCR or RNA-sequencing).
Research team and the project management
The team consists of 10 people: Doctor of Science – 1; PhD – 6; PhD students – 2; masters – 1. All scientists will be 100% employed during the entire period of the project.