Title page for ETD etd-11172005-162404


Type of Document Dissertation
Author Roche, Rebecca I.
URN etd-11172005-162404
Title Role of RNA Processing Factors in the Expression of Flt-1 and its Secreted Variant, sFlt-1
Degree PhD
Department Veterinary Medical Sciences
Advisory Committee
Advisor Name Title
Huckle, William R. Committee Chair
Eyestone, Willard H. Committee Member
Gillaspy, Glenda E. Committee Member
Meng, Xiang-Jin Committee Member
Robertson, John L. Committee Member
Sible, Jill C. Committee Member
Keywords
  • Vascular Endothelial Growth Factor
  • Angiogenesis
Date of Defense 2005-11-09
Availability unrestricted
Abstract
Role of RNA Processing Factors in the Expression of Flt-1

and its Secreted Variant, sFlt-1

By

Rebecca I. Roche, MS

Dr. W.R. Huckle, Chairman

Department of Biomedical Sciences and Pathobiology

(ABSTRACT)

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.

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