Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen involved in angiogenesis, the formation of new blood vessels. sFlt-1, a secreted form of the signal-transducing VEGF receptor Flt-1, can inhibit cellular responses to VEGF both in vitro and in vivo. sFlt-1 is generated by alternative pre-mRNA processing; removal of Flt-1 intron 13 by splicing produces the mRNA for transmembrane Flt-1, whereas cleavage/polyadenylation within this intron, preserving the exon 13/intron 13 junction, yields sFlt-1 mRNA. Despite the likely importance of sFlt-1 in VEGF signaling, little is known about the regulation of its expression.
Previous studies using an Flt-1 minigene (pFIN13) revealed that intronic cleavage/polyadenylation signals can affect Flt-1 expression, and, conversely, that 3' intronic splice signals can affect sFlt-1 expression. The goal of present work was to test the hypothesis that splicing and cleavage/polyadenylation factors compete functionally on Flt-1 transcripts, by 1) assessing the influence of exon 13/14 splicing determinants on expression of Flt-1 RNA processing variants in a transfected cell model system; 2) determining the effects of altering the relative abundance of proteins principally involved in splicing or cleavage/polyadenylation; and 3) characterizing a previously-unknown splice variant, predicted to encode a novel sFlt-1 protein isoform, in cells overexpressing the spliceosomal RNA binding protein U2AF65.
When the upstream exon in pFIN13 was decreased from 2135 to 309 bp, the sFlt-1:Flt-1 mRNA ratio decreased 8.9-fold and an aberrant 5'UTR/exon 14 splice decreased 60-fold, indicating that "exon definition" is a key parameter of successful Flt-1 RNA processing. Mutation of 5' or 3' intronic splice signals had little effect on Long sFlt-1:Total sFlt-1 mRNA ratio, suggesting that splicing and cleavage/polyadenylation factors may not compete physically for Flt-1 transcripts. Although co-transfection with RNA processing factor cDNAs did not generally produce the predicted pattern of effects on sFlt-1:Flt-1 mRNA ratio, a cryptic exon within intron 13 was revealed in cells overexpressing U2AF65. sFlt-1 protein apparently can be encoded by mRNAs either cleaved/polyadenylated within intron 13 or, surprisingly, by splicing of the cryptic exon "13b." Thus, the cellular decision to produce sFlt-1 or Flt-1 from a nascent RNA can no longer be viewed as a simple choice between cleavage/polyadenylation and splicing. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/29632 |
| Date | 21 November 2005 |
| Creators | Roche, Rebecca I. |
| Contributors | Veterinary Medical Sciences, Huckle, William R., Sible, Jill C., Meng, Xiang-Jin, Robertson, John L., Gillaspy, Glenda E., Eyestone, Willard H. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | ROCHE_Dissertation_etdREV.pdf |
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