Identification of an exonic splicing silencer in exon 6A of the human VEGF gene

Rui Wang, Ronald Crystal, Neil R. Hackett

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Background: 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. Results: 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. Conclusion: This work establishes the identity of cis sequences that regulate alternative VEGF splicing and dictate the relative expression levels of VEGF isoforms.

Original languageEnglish
Article number103
JournalBMC Molecular Biology
Volume10
DOIs
Publication statusPublished - 17 Nov 2009
Externally publishedYes

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Exons
Vascular Endothelial Growth Factor A
Genes
Protein Isoforms
Plasmids
Messenger RNA
Consensus Sequence
Alternative Splicing
human VEGFA protein
Mutagenesis
Blood Vessels
Nucleotides
Growth

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Identification of an exonic splicing silencer in exon 6A of the human VEGF gene. / Wang, Rui; Crystal, Ronald; Hackett, Neil R.

In: BMC Molecular Biology, Vol. 10, 103, 17.11.2009.

Research output: Contribution to journalArticle

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