September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
RNA-Sequencing data supports the existence of novel VEGFA splicing events but not of VEGFxxxb isoforms
Author Affiliations & Notes
  • David A Simpson
    Centre for Experimental Medicine, Queens University Belfast, Belfast, Northern Ireland, United Kingdom
  • Stephen Bridgett
    Centre for Experimental Medicine, Queens University Belfast, Belfast, Northern Ireland, United Kingdom
  • Margaret Dellett
    Centre for Experimental Medicine, Queens University Belfast, Belfast, Northern Ireland, United Kingdom
  • Footnotes
    Commercial Relationships   David Simpson, None; Stephen Bridgett, None; Margaret Dellett, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4785. doi:
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      David A Simpson, Stephen Bridgett, Margaret Dellett; RNA-Sequencing data supports the existence of novel VEGFA splicing events but not of VEGFxxxb isoforms. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4785.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Vascular endothelial growth factor (VEGFA) is a pivotal regulator of angiogenesis and valuable therapeutic target for neovascular eye diseases. Three principal isoforms, VEGF121, VEGF165 and VEGF189 are generated by alternative splicing. Anti-angiogenic ‘VEGFxxxb’ isoforms that utilise an alternative splice site in the final exon detected by RT-PCR have been widely reported. These have enormous potential functional implications, but their existence has been questioned. We therefore sought novel splice isoforms and confirmation of the existence of the VEGFxxxb isoforms within RNA sequencing data.

Methods : Publicly available RNA-sequencing data from a range of tissues was downloaded from Arrayexpress (E-MTAB-513; E-MTAB-1733). Reads were aligned to chromosome 6, including the VEGFA gene using the STAR aligner and the number of reads matching specific splice junctions identified by BLAST. RT-PCR spanning exon 7-8 was performed on RNA from multiple tissues and the products sequenced on an Ion Torrent PGM. Retinal transcriptome data from the Ocular Genomics Institute , Harvard was also analysed.

Results : Amongst a total of more than 7 billion reads from 35 tissues, 45,000 reads included exon 8 splice junctions from the canonical VEGFA isoforms but there were no sequences derived from VEGFxxxb isoforms. Sequencing of approximately 50,000 RT-PCR products spanning the exon7-8 junction in 10 tissues did not identify any VEGFxxxb transcripts. No VEGFxxxB splice junctions were identified in retinal RNA. Our analyses also revealed multiple novel splicing events supported by more reads than previously reported VEGF-145 and -148 isoforms. These included several from novel first exons which were supported by existing transcription start site data and are predicted to encode isoforms lacking a signal peptide. One of these isoforms is present in RNA-Seq data from human retinal RNA.

Conclusions : Although the absence or extremely low expression of VEGFxxxb transcripts in vivo indicates that these isoforms do not play a role in normal physiology, the published observations of functional effects following overexpression of artificial VEGFxxxb isoforms remain extremely valuable. The novel VEGFA isoforms identified may play significant roles in specific cell types, including in the retina.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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