Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo

Jason A. Somarelli, Daneen Schaeffer, Reggie Bosma, Vivian I. Bonano, Jang Wook Sohn, Gabor Kemeny, Abhinav Ettyreddy, Mariano A. Garcia-Blanco

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Alternative splicing generates a vast diversity of protein isoforms from a limited number of protein-coding genes, with many of the isoforms possessing unique, and even contrasting, functions. Fluorescence-based splicing reporters have the potential to facilitate studies of alternative splicing at the single-cell level and can provide valuable information on phenotypic transitions in almost real time. Fibroblast growth factor receptor 2 (FGFR2) pre-mRNA is alternatively spliced to form the epithelial-specific and mesenchymal-specific IIIb and IIIc isoforms, respectively, which are useful markers of epithelial-mesenchymal transitions (EMT). We have used our knowledge of FGFR2 splicing regulation to develop a fluorescence-based reporter system to visualize exon IIIc regulation in vitro and in vivo. Here we show the application of this reporter system to the study of EMT in vitro in cell culture and in vivo in transgenic mice harboring these splicing constructs. In explant studies, the reporters revealed that FGFR2 isoform switching is not required for keratinocyte migration during cutaneous wound closure. Our results demonstrate the value of the splicing reporters as tools to study phenotypic transitions and cell fates at single cell resolution. Moreover, our data suggest that keratinocytes migrate efficiently in the absence of a complete EMT.

Original languageEnglish
Pages (from-to)116-127
Number of pages12
JournalRNA
Volume19
Issue number1
DOIs
Publication statusPublished - 2013 Jan 1

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Receptor, Fibroblast Growth Factor, Type 2
Alternative Splicing
Epithelial-Mesenchymal Transition
Protein Isoforms
Fluorescence
Keratinocytes
RNA Precursors
Transgenic Mice
Exons
Cell Culture Techniques
Skin
Wounds and Injuries
Proteins
In Vitro Techniques

Keywords

  • DsRed
  • Enhanced green fluorescent protein
  • Epithelial-mesenchymal transition
  • In vivo imaging
  • pre-mRNA splicing
  • Wound healing

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Somarelli, J. A., Schaeffer, D., Bosma, R., Bonano, V. I., Sohn, J. W., Kemeny, G., ... Garcia-Blanco, M. A. (2013). Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo. RNA, 19(1), 116-127. https://doi.org/10.1261/rna.035097.112

Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo. / Somarelli, Jason A.; Schaeffer, Daneen; Bosma, Reggie; Bonano, Vivian I.; Sohn, Jang Wook; Kemeny, Gabor; Ettyreddy, Abhinav; Garcia-Blanco, Mariano A.

In: RNA, Vol. 19, No. 1, 01.01.2013, p. 116-127.

Research output: Contribution to journalArticle

Somarelli, JA, Schaeffer, D, Bosma, R, Bonano, VI, Sohn, JW, Kemeny, G, Ettyreddy, A & Garcia-Blanco, MA 2013, 'Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo', RNA, vol. 19, no. 1, pp. 116-127. https://doi.org/10.1261/rna.035097.112
Somarelli, Jason A. ; Schaeffer, Daneen ; Bosma, Reggie ; Bonano, Vivian I. ; Sohn, Jang Wook ; Kemeny, Gabor ; Ettyreddy, Abhinav ; Garcia-Blanco, Mariano A. / Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo. In: RNA. 2013 ; Vol. 19, No. 1. pp. 116-127.
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