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Alternate processing of Flt1 transcripts is directed by conserved cis-elements within an intronic region of FLT1 that reciprocally regulates splicing and polyadenylation.

Thomas CP, Raikwar NS, Kelley EA, Liu KZ - Nucleic Acids Res. (2010)

Bottom Line: Phorbol myristic acid and dimethyloxalylglycine differentially stimulate sFlt1 compared to Flt1 expression in vascular endothelial cells and in cytotrophoblasts.Inclusion of exon 15 but not 14 had a modest stimulatory effect on the abundance of sFlt1.We conclude that intronic signals reciprocally regulate splicing and polyadenylation and control sFlt1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA, USA. christie-thomas@uiowa.edu

ABSTRACT
The vascular endothelial growth factor receptor, Flt1 is a transmembrane receptor co-expressed with an alternate transcript encoding a secreted form, sFlt1, that functions as a competitive inhibitor of Flt1. Despite shared transcription start sites and upstream regulatory elements, sFlt1 is in far greater excess of Flt1 in the human placenta. Phorbol myristic acid and dimethyloxalylglycine differentially stimulate sFlt1 compared to Flt1 expression in vascular endothelial cells and in cytotrophoblasts. An FLT1 minigene construct containing exon 13, 14 and the intervening region, recapitulates mRNA processing when transfected into COS-7, with chimeric intronic sFlt1 transcripts arising by intronic polyadenylation and other Flt1/sFlt1 transcripts by alternate splicing. Inclusion of exon 15 but not 14 had a modest stimulatory effect on the abundance of sFlt1. The intronic region containing the distal poly(A) signal sequences, when transferred to a heterologous minigene construct, inhibited splicing but only when cloned in sense orientation, consistent with the presence of a directional cis-element. Serial deletional and targeted mutational analysis of cis-elements within intron 13 identified intronic poly(A) signal sequences and adjacent cis-elements as the principal determinants of the relative ratio of intronic sFlt1 and spliced Flt1. We conclude that intronic signals reciprocally regulate splicing and polyadenylation and control sFlt1 expression.

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Mapping cis-elements within intron 13 that regulate the relative abundance of intronic sFlt1 and Flt1. (A) Schematic representation of human FLT1 gene in the region of exons 13 and 14. Exon 13 splices to exon 14 to form Flt1; alternatively, intronic polyadenylation sites are used to create a composite 3′ terminal exon for sFlt1. The position of the putative proximal poly(A) signal sequence, the known distal poly(A) signal sequences and the restriction sites used to map cis-elements are shown. (B) RPA demonstrating relative abundance of intronic sFlt1 and spliced Flt1 in COS-7 cells transfected with a series of Flt1 minigene constructs. The full-length construct leads primarily to intronic sFlt1, and the largest deletions leads primarily to spliced Flt1. Progressive 5′–3′ deletions lead to a fall in processed Flt1. (C) Pooled quantitated data from several experiments with the Flt1 to sFlt1 ratio in each condition expressed as fold increase compared to the ratio in the full-length construct. n = 3, mean ± SEM, *P < 0.01, ANOVA (by ranks).
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Figure 3: Mapping cis-elements within intron 13 that regulate the relative abundance of intronic sFlt1 and Flt1. (A) Schematic representation of human FLT1 gene in the region of exons 13 and 14. Exon 13 splices to exon 14 to form Flt1; alternatively, intronic polyadenylation sites are used to create a composite 3′ terminal exon for sFlt1. The position of the putative proximal poly(A) signal sequence, the known distal poly(A) signal sequences and the restriction sites used to map cis-elements are shown. (B) RPA demonstrating relative abundance of intronic sFlt1 and spliced Flt1 in COS-7 cells transfected with a series of Flt1 minigene constructs. The full-length construct leads primarily to intronic sFlt1, and the largest deletions leads primarily to spliced Flt1. Progressive 5′–3′ deletions lead to a fall in processed Flt1. (C) Pooled quantitated data from several experiments with the Flt1 to sFlt1 ratio in each condition expressed as fold increase compared to the ratio in the full-length construct. n = 3, mean ± SEM, *P < 0.01, ANOVA (by ranks).

Mentions: We then created a series of deletion constructs to identify cis-elements within intron 13 that regulate processing of Flt1. In contrast to the full-length construct, deletion of a ∼4300 bp SmaI–NheI intronic fragment switched processing almost completely to spliced ‘Flt1’, clearly demonstrating that cis-elements within intron 13 regulate both polyadenylation and splicing (Figure 3A–C). The shift from intronic polyadenylation to splicing was not from loss of all polyadenylation signal sequences since the proximal sFlt1 polyA signal sequence is still present in the largest deletion construct. We demonstrate that as the deleted region gets progressively smaller in a 5′–3′ direction, there is progressively less splicing to Flt1 (Figure 3B and C). This suggests that there are cooperative or additive interactions between upstream cis-elements and the distal polyA sites that promote processing to intronic sFlt1 and that the loss of the distal polyA sites was still sufficient to stimulate modest splicing of exon 13 to exon 14.Figure 3.


Alternate processing of Flt1 transcripts is directed by conserved cis-elements within an intronic region of FLT1 that reciprocally regulates splicing and polyadenylation.

Thomas CP, Raikwar NS, Kelley EA, Liu KZ - Nucleic Acids Res. (2010)

Mapping cis-elements within intron 13 that regulate the relative abundance of intronic sFlt1 and Flt1. (A) Schematic representation of human FLT1 gene in the region of exons 13 and 14. Exon 13 splices to exon 14 to form Flt1; alternatively, intronic polyadenylation sites are used to create a composite 3′ terminal exon for sFlt1. The position of the putative proximal poly(A) signal sequence, the known distal poly(A) signal sequences and the restriction sites used to map cis-elements are shown. (B) RPA demonstrating relative abundance of intronic sFlt1 and spliced Flt1 in COS-7 cells transfected with a series of Flt1 minigene constructs. The full-length construct leads primarily to intronic sFlt1, and the largest deletions leads primarily to spliced Flt1. Progressive 5′–3′ deletions lead to a fall in processed Flt1. (C) Pooled quantitated data from several experiments with the Flt1 to sFlt1 ratio in each condition expressed as fold increase compared to the ratio in the full-length construct. n = 3, mean ± SEM, *P < 0.01, ANOVA (by ranks).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2926593&req=5

Figure 3: Mapping cis-elements within intron 13 that regulate the relative abundance of intronic sFlt1 and Flt1. (A) Schematic representation of human FLT1 gene in the region of exons 13 and 14. Exon 13 splices to exon 14 to form Flt1; alternatively, intronic polyadenylation sites are used to create a composite 3′ terminal exon for sFlt1. The position of the putative proximal poly(A) signal sequence, the known distal poly(A) signal sequences and the restriction sites used to map cis-elements are shown. (B) RPA demonstrating relative abundance of intronic sFlt1 and spliced Flt1 in COS-7 cells transfected with a series of Flt1 minigene constructs. The full-length construct leads primarily to intronic sFlt1, and the largest deletions leads primarily to spliced Flt1. Progressive 5′–3′ deletions lead to a fall in processed Flt1. (C) Pooled quantitated data from several experiments with the Flt1 to sFlt1 ratio in each condition expressed as fold increase compared to the ratio in the full-length construct. n = 3, mean ± SEM, *P < 0.01, ANOVA (by ranks).
Mentions: We then created a series of deletion constructs to identify cis-elements within intron 13 that regulate processing of Flt1. In contrast to the full-length construct, deletion of a ∼4300 bp SmaI–NheI intronic fragment switched processing almost completely to spliced ‘Flt1’, clearly demonstrating that cis-elements within intron 13 regulate both polyadenylation and splicing (Figure 3A–C). The shift from intronic polyadenylation to splicing was not from loss of all polyadenylation signal sequences since the proximal sFlt1 polyA signal sequence is still present in the largest deletion construct. We demonstrate that as the deleted region gets progressively smaller in a 5′–3′ direction, there is progressively less splicing to Flt1 (Figure 3B and C). This suggests that there are cooperative or additive interactions between upstream cis-elements and the distal polyA sites that promote processing to intronic sFlt1 and that the loss of the distal polyA sites was still sufficient to stimulate modest splicing of exon 13 to exon 14.Figure 3.

Bottom Line: Phorbol myristic acid and dimethyloxalylglycine differentially stimulate sFlt1 compared to Flt1 expression in vascular endothelial cells and in cytotrophoblasts.Inclusion of exon 15 but not 14 had a modest stimulatory effect on the abundance of sFlt1.We conclude that intronic signals reciprocally regulate splicing and polyadenylation and control sFlt1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA, USA. christie-thomas@uiowa.edu

ABSTRACT
The vascular endothelial growth factor receptor, Flt1 is a transmembrane receptor co-expressed with an alternate transcript encoding a secreted form, sFlt1, that functions as a competitive inhibitor of Flt1. Despite shared transcription start sites and upstream regulatory elements, sFlt1 is in far greater excess of Flt1 in the human placenta. Phorbol myristic acid and dimethyloxalylglycine differentially stimulate sFlt1 compared to Flt1 expression in vascular endothelial cells and in cytotrophoblasts. An FLT1 minigene construct containing exon 13, 14 and the intervening region, recapitulates mRNA processing when transfected into COS-7, with chimeric intronic sFlt1 transcripts arising by intronic polyadenylation and other Flt1/sFlt1 transcripts by alternate splicing. Inclusion of exon 15 but not 14 had a modest stimulatory effect on the abundance of sFlt1. The intronic region containing the distal poly(A) signal sequences, when transferred to a heterologous minigene construct, inhibited splicing but only when cloned in sense orientation, consistent with the presence of a directional cis-element. Serial deletional and targeted mutational analysis of cis-elements within intron 13 identified intronic poly(A) signal sequences and adjacent cis-elements as the principal determinants of the relative ratio of intronic sFlt1 and spliced Flt1. We conclude that intronic signals reciprocally regulate splicing and polyadenylation and control sFlt1 expression.

Show MeSH
Related in: MedlinePlus