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

Rui Wang,Neil R Hackett,Ronald G Crystal

doi:10.1186/1471-2199-10-103

Abstract

BackgroundThe 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.ResultsA 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.ConclusionThis work establishes the identity of cis sequences that regulate alternative VEGF splicing and dictate the relative expression levels of VEGF isoforms.

Highlights

The different isoforms of vascular endothelial growth factor (VEGF) play diverse roles in vascular growth, structure and function
The minigene consisted of a green fluorescent protein (GFP) expression plasmid in which the GFP gene sequence was interrupted by a shortened version of intron 5, the complete exon 6A and a shortened version of intron 6 all from the human VEGF gene (Figure 1)
If the splicing pathway corresponds to that of VEGF189, exon 6A will be included in the final mRNA and the GFP open reading

Summary

Introduction

The different isoforms of vascular endothelial growth factor (VEGF) play diverse roles in vascular growth, structure and function. At least eight different isoforms of VEGF are formed, comprising VEGF206, 189, 183, 165, 148, 145, 121, and an inhibitory isoforms, 165b [11,12,13]. VEGF189 mRNA is relatively abundant in mouse lung and heart, but VEGF165 mRNA has the highest level of expression in most other mouse tissues [17,18,19,20]. These two isoforms differ by the presence or absence of exon 6A which encodes the critical (page number not for citation purposes)

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Conclusion