{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/29632"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/29632","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Role of RNA Processing Factors in the Expression of Flt-1 and its Secreted Variant, sFlt-1","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Roche, Rebecca I."],"institution":"Virginia Tech","degree_name":"Ph. D.","degree_level":"doctoral","degree_discipline":"Veterinary Medical Sciences","degree_department":"Veterinary Medical Sciences","school":null,"contributors":[],"advisors":[],"committee_chairs":["Huckle, William R."],"committee_members":["Sible, Jill C.","Meng, Xiang-Jin","Robertson, John L.","Gillaspy, Glenda E.","Eyestone, Willard H."],"year":2005,"date_issued":"2005-11-09","date_published":"2005-11-09","updated_at":"2026-07-22T22:18:50Z","subjects":["Vascular Endothelial Growth Factor","Angiogenesis"],"languages":[],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-11172005-162404"],"render_values":[{"text":"etd-11172005-162404","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/29632","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Huckle, William R."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Sible, Jill C.","Meng, Xiang-Jin","Robertson, John L.","Gillaspy, Glenda E.","Eyestone, Willard H."]},{"key":"dc:contributor.department","label":"Department","values":["Veterinary Medical Sciences"]},{"key":"dc:creator","label":"Author","values":["Roche, Rebecca I."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2014-03-14T20:18:40Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2014-03-14T20:18:40Z","2006-11-21"]},{"key":"dc:date.issued","label":"Date","values":["2005-11-09"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Veterinary Medical Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph. 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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."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph. D."]},{"key":"dc:title","label":"Title","values":["Role of RNA Processing Factors in the Expression of Flt-1 and its Secreted Variant, sFlt-1"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Huckle, William R."],"dc:contributor.committeemember":["Sible, Jill C.","Meng, Xiang-Jin","Robertson, John L.","Gillaspy, Glenda E.","Eyestone, Willard H."],"dc:contributor.department":["Veterinary Medical Sciences"],"dc:creator":["Roche, Rebecca I."],"dc:date.accessioned":["2014-03-14T20:18:40Z"],"dc:date.available":["2014-03-14T20:18:40Z","2006-11-21"],"dc:date.issued":["2005-11-09"],"dc:description.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."],"dc:description.degree":["Ph. D."],"dc:identifier.other":["etd-11172005-162404"],"dc:identifier.uri":["http://hdl.handle.net/10919/29632"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["Vascular Endothelial Growth Factor","Angiogenesis"],"dc:title":["Role of RNA Processing Factors in the Expression of Flt-1 and its Secreted Variant, sFlt-1"],"dc:type":["Dissertation"],"thesis:degree_discipline":["Veterinary Medical Sciences"],"thesis:degree_level":["doctoral"],"thesis:degree_name":["Ph. D."],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:18:50Z"}