Identification of an exonic splicing silencer in exon 6A of the human VEGF gene. Academic Article uri icon

Overview

MeSH

  • Base Sequence
  • Genes, Reporter
  • Genome, Human
  • Green Fluorescent Proteins
  • Humans
  • Mutagenesis
  • Reproducibility of Results
  • Sequence Deletion
  • Transfection

MeSH Major

  • Exons
  • RNA Splicing
  • Silencer Elements, Transcriptional
  • Vascular Endothelial Growth Factor A

abstract

  • The different isoforms of vascular endothelial growth factor (VEGF) play diverse roles in vascular growth, structure and function. Alternative splicing of the VEGF gene results in the expression of three abundant isoforms: VEGF121, VEGF165 and VEGF189. The mRNA for VEGF189 contains the alternatively spliced exon 6A whereas the mRNA for VEGF165 lacks this exon. The objective of this study was to identify the cis elements that control utilization of exon 6A. A reporter minigene was constructed (pGFP-E6A) containing the coding sequence for GFP whose translation was dependent on faithful splicing for removal of the VEGF exon 6A. To identify cis-acting splicing elements, sequential deletions were made across exon 6A in the pGFP-E6A plasmid. A candidate cis-acting exonic splicing silencer (ESS) comprising nucleotides 22-30 of exon 6A sequence was identified corresponding to the a silencer consensus sequence of AAGGGG. The function of this sequence as an ESS was confirmed in vivo both in the context of the reporter minigene as a plasmid and in the context of a longer minigene with VEGF exon 6A in its native context in an adenoviral gene transfer vector. Further mutagenesis studies resulted in the identification of the second G residue of the putative ESS as the most critical for function. This work establishes the identity of cis sequences that regulate alternative VEGF splicing and dictate the relative expression levels of VEGF isoforms.

publication date

  • 2009

has subject area

  • Base Sequence
  • Exons
  • Genes, Reporter
  • Genome, Human
  • Green Fluorescent Proteins
  • Humans
  • Mutagenesis
  • RNA Splicing
  • Reproducibility of Results
  • Sequence Deletion
  • Silencer Elements, Transcriptional
  • Transfection
  • Vascular Endothelial Growth Factor A

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC2784459

Digital Object Identifier (DOI)

  • 10.1186/1471-2199-10-103

PubMed ID

  • 19922608

Additional Document Info

start page

  • 103

volume

  • 10