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UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts

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Abstract
Background: The complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation through interaction with RNA. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo-dT beads and identifying the crosslinked proteins using mass spectrometry. We describe here an optimized system of mRNA interactome capture for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type often used in functional cellular assays. Results: We established the conditions for optimal protein yield, namely the amount of starting tissue, the duration of UV irradiation and the effect of UV intensity. We demonstrated high efficiency mRNA-protein pull-down by oligo-d(T)(25) bead capture. Proteins annotated to have RNA-binding capacity were overrepresented in the obtained medium scale mRNA-bound proteome, indicating the specificity of the method and providing in vivo UV crosslinking experimental evidence for several candidate RBPs from leaf mesophyll protoplasts. Conclusions: The described method, applied to plant cells, allows identifying proteins as having the capacity to bind mRNA directly. The method can now be scaled and applied to other plant cell types and species to contribute to the comprehensive description of the RBP proteome of plants.
Keywords
Messenger RNA-binding proteins, Messenger ribonucleoprotein complexes, Arabidopsis thaliana leaf mesophyll protoplasts, In vivo UV crosslinking, mRNA-bound proteome, TRANSIENT GENE-EXPRESSION, POLY(A)-BINDING PROTEIN, MASS-SPECTROMETRY, ARABIDOPSIS-THALIANA, FLOWERING TIME, LINKING, RECOGNITION, RESOLUTION, YEAST, IDENTIFICATION

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MLA
Zhang, Zhicheng et al. “UV Crosslinked mRNA-binding Proteins Captured from Leaf Mesophyll Protoplasts.” PLANT METHODS 12 (2016): n. pag. Print.
APA
Zhang, Zhicheng, Boonen, K., Ferrari, P., Schoofs, L., Janssens, E., van Noort, V., Rolland, F., et al. (2016). UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts. PLANT METHODS, 12.
Chicago author-date
Zhang, Zhicheng, Kurt Boonen, Piero Ferrari, Liliane Schoofs, Ewald Janssens, Vera van Noort, Filip Rolland, and Koen Geuten. 2016. “UV Crosslinked mRNA-binding Proteins Captured from Leaf Mesophyll Protoplasts.” Plant Methods 12.
Chicago author-date (all authors)
Zhang, Zhicheng, Kurt Boonen, Piero Ferrari, Liliane Schoofs, Ewald Janssens, Vera van Noort, Filip Rolland, and Koen Geuten. 2016. “UV Crosslinked mRNA-binding Proteins Captured from Leaf Mesophyll Protoplasts.” Plant Methods 12.
Vancouver
1.
Zhang Z, Boonen K, Ferrari P, Schoofs L, Janssens E, van Noort V, et al. UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts. PLANT METHODS. 2016;12.
IEEE
[1]
Z. Zhang et al., “UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts,” PLANT METHODS, vol. 12, 2016.
@article{8544961,
  abstract     = {Background: The complexity of RNA regulation is one of the current frontiers in animal and plant molecular biology research. RNA-binding proteins (RBPs) are characteristically involved in post-transcriptional gene regulation through interaction with RNA. Recently, the mRNA-bound proteome of mammalian cell lines has been successfully cataloged using a new method called interactome capture. This method relies on UV crosslinking of proteins to RNA, purifying the mRNA using complementary oligo-dT beads and identifying the crosslinked proteins using mass spectrometry. We describe here an optimized system of mRNA interactome capture for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type often used in functional cellular assays. 
Results: We established the conditions for optimal protein yield, namely the amount of starting tissue, the duration of UV irradiation and the effect of UV intensity. We demonstrated high efficiency mRNA-protein pull-down by oligo-d(T)(25) bead capture. Proteins annotated to have RNA-binding capacity were overrepresented in the obtained medium scale mRNA-bound proteome, indicating the specificity of the method and providing in vivo UV crosslinking experimental evidence for several candidate RBPs from leaf mesophyll protoplasts. 
Conclusions: The described method, applied to plant cells, allows identifying proteins as having the capacity to bind mRNA directly. The method can now be scaled and applied to other plant cell types and species to contribute to the comprehensive description of the RBP proteome of plants.},
  articleno    = {42},
  author       = {Zhang, Zhicheng and Boonen, Kurt and Ferrari, Piero and Schoofs, Liliane and Janssens, Ewald and van Noort, Vera and Rolland, Filip and Geuten, Koen},
  issn         = {1746-4811},
  journal      = {PLANT METHODS},
  keywords     = {Messenger RNA-binding proteins,Messenger ribonucleoprotein complexes,Arabidopsis thaliana leaf mesophyll protoplasts,In vivo UV crosslinking,mRNA-bound proteome,TRANSIENT GENE-EXPRESSION,POLY(A)-BINDING PROTEIN,MASS-SPECTROMETRY,ARABIDOPSIS-THALIANA,FLOWERING TIME,LINKING,RECOGNITION,RESOLUTION,YEAST,IDENTIFICATION},
  language     = {eng},
  pages        = {12},
  title        = {UV crosslinked mRNA-binding proteins captured from leaf mesophyll protoplasts},
  url          = {http://dx.doi.org/10.1186/s13007-016-0142-6},
  volume       = {12},
  year         = {2016},
}

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