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mRNA interactome capture from plant protoplasts

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Abstract
The complexity of interactions between RNA and regulatory RNA-binding proteins (RBPs) contributes to RNA regulation being one of the main frontiers in molecular biology. RNA-binding proteins mainly participate in the various RNA biogenesis pathways, especially post-transcriptional gene regulation of messenger RNAs (mRNAs). Recently, the mRNA-bound proteomes of yeast and mammalian cell lines have been isolated using a novel method called "interactome capture", which allowed to comprehensively unravel and catalog functional mRBPs from a physiological cellular environment. In comparison, the experimental evidence for interactions in the plant mRNA-bound proteome is still limited making studies of plant RBPs timely. We present an optimized system of mRNA interactome capture for plant protoplasts and apply this to Arabidopsis thaliana leaf mesophyll protoplasts, a cell type used in functional cellular assays. Similar to the developed interactome capture for yeast and mammalian cells, this method relies on in vivo UV crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo-dT beads and subsequent identification of the crosslinked proteins by mass spectrometry (MS). The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the obtained medium scale mRNA-bound proteome, previously annotated RBPs with RNA-binding capacity are overrepresented and novel RBPs are identified. The method can be scaled and applied to other plant cell types and species to broadly discover, catalog and compare mRNA-bound proteomes in plants.

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MLA
Zhang, Zhicheng, Kurt Boonen, Meixia Li, et al. “mRNA Interactome Capture from Plant Protoplasts.” JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 125 (2017): n. pag. Print.
APA
Zhang, Zhicheng, Boonen, K., Li, M., & Geuten, K. (2017). mRNA interactome capture from plant protoplasts. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, (125).
Chicago author-date
Zhang, Zhicheng, Kurt Boonen, Meixia Li, and Koen Geuten. 2017. “mRNA Interactome Capture from Plant Protoplasts.” Jove-journal of Visualized Experiments (125).
Chicago author-date (all authors)
Zhang, Zhicheng, Kurt Boonen, Meixia Li, and Koen Geuten. 2017. “mRNA Interactome Capture from Plant Protoplasts.” Jove-journal of Visualized Experiments (125).
Vancouver
1.
Zhang Z, Boonen K, Li M, Geuten K. mRNA interactome capture from plant protoplasts. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS. 2017;(125).
IEEE
[1]
Z. Zhang, K. Boonen, M. Li, and K. Geuten, “mRNA interactome capture from plant protoplasts,” JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, no. 125, 2017.
@article{8544962,
  abstract     = {The complexity of interactions between RNA and regulatory RNA-binding proteins (RBPs) contributes to RNA regulation being one of the main frontiers in molecular biology. RNA-binding proteins mainly participate in the various RNA biogenesis pathways, especially post-transcriptional gene regulation of messenger RNAs (mRNAs). Recently, the mRNA-bound proteomes of yeast and mammalian cell lines have been isolated using a novel method called "interactome capture", which allowed to comprehensively unravel and catalog functional mRBPs from a physiological cellular environment. In comparison, the experimental evidence for interactions in the plant mRNA-bound proteome is still limited making studies of plant RBPs timely. We present an optimized system of mRNA interactome capture for plant protoplasts and apply this to Arabidopsis thaliana leaf mesophyll protoplasts, a cell type used in functional cellular assays. Similar to the developed interactome capture for yeast and mammalian cells, this method relies on in vivo UV crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo-dT beads and subsequent identification of the crosslinked proteins by mass spectrometry (MS). The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the obtained medium scale mRNA-bound proteome, previously annotated RBPs with RNA-binding capacity are overrepresented and novel RBPs are identified. The method can be scaled and applied to other plant cell types and species to broadly discover, catalog and compare mRNA-bound proteomes in plants.},
  articleno    = {e56011},
  author       = {Zhang, Zhicheng and Boonen, Kurt and Li, Meixia and Geuten, Koen},
  issn         = {1940-087X},
  journal      = {JOVE-JOURNAL OF VISUALIZED EXPERIMENTS},
  language     = {eng},
  number       = {125},
  title        = {mRNA interactome capture from plant protoplasts},
  url          = {http://dx.doi.org/10.3791/56011},
  year         = {2017},
}

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