14-3-3 isoforms bind directly exon B of the 5ʹ-UTR of human surfactant protein A2 mRNA

Georgios T. Noutsios, Paul Ghattas, Stephanie Bennett, Joanna Floros

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5ʹ- untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722–L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5ʹ-UTR mRNA.

Original languageEnglish (US)
Pages (from-to)L147-L157
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume309
Issue number2
DOIs
StatePublished - Jul 15 2015

Keywords

  • 14- 3-3
  • 5ʹ-untranslated region
  • Human pulmonary surfactant protein A
  • Translation

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

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