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mRNA Interactome Capture from Plant Protoplasts
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mRNA Interactome Capture from Plant Protoplasts.

Zhicheng Zhang1, Kurt Boonen1, Meixia Li1

  • 1Department of Biology, KU Leuven.

Journal of Visualized Experiments : Jove
|August 9, 2017
PubMed
Summary

Researchers optimized a novel mRNA interactome capture method to identify RNA-binding proteins (RBPs) in Arabidopsis thaliana leaf cells. This method aids in understanding post-transcriptional gene regulation in plants.

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Area of Science:

  • Molecular Biology
  • Plant Science
  • Biochemistry

Background:

  • RNA-binding proteins (RBPs) are crucial regulators of RNA biology, influencing all RNA biogenesis pathways and post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs).
  • The mRNA interactome capture method has enabled the identification of mRNA-binding proteins (mRBPs) in yeast and mammalian cells by isolating messenger ribonucleoprotein complexes (mRNPs).
  • Recent studies have applied this method to various Arabidopsis thaliana tissues, highlighting its potential for plant research.

Purpose of the Study:

  • To optimize the mRNA interactome capture method specifically for Arabidopsis thaliana leaf mesophyll protoplasts.
  • To improve protein yield and identify novel RBPs within these plant cells.
  • To establish a scalable and broadly applicable method for cataloging plant mRNA-bound proteomes.

Main Methods:

  • In vivo ultraviolet (UV) crosslinking of cells.
  • Pull-down and purification of mRNPs using oligo(dT) beads.
  • Identification of crosslinked proteins via mass spectrometry (MS).

Main Results:

  • Optimized conditions for protein yield were determined, focusing on starting tissue amount and UV irradiation duration.
  • In a medium-scale experiment, RBPs with known RNA-binding capacity were significantly overrepresented.
  • Numerous novel RBPs were identified in the Arabidopsis leaf mesophyll protoplast proteome.
  • The method demonstrated scalability for large-scale experiments (up to 10^9 cells).

Conclusions:

  • The optimized mRNA interactome capture method provides an efficient means to isolate and identify mRBPs in Arabidopsis thaliana leaf mesophyll protoplasts.
  • This technique facilitates a deeper understanding of PTGR in plants.
  • The optimized method is adaptable for diverse plant cell types and species, enabling comprehensive proteome-wide studies.