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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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The AflIII–NheI region of FLT1 intron 13 is required to significantly reduce splicing of exon 13 to exon 14. (A) Schematic representation of human FLT1 gene in the region of exons 13 and 14. 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 is 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 deletions that do not remove the distal polyA sites do not substantially increase the abundance of spliced Flt1. Deletion of the region between AflIII and NheI, has a significant effect on processing to spliced 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 = 4, mean ± SEM, P < 0.001, ANOVA, *P < 0.05 compared to each of the other samples.
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Figure 4: The AflIII–NheI region of FLT1 intron 13 is required to significantly reduce splicing of exon 13 to exon 14. (A) Schematic representation of human FLT1 gene in the region of exons 13 and 14. 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 is 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 deletions that do not remove the distal polyA sites do not substantially increase the abundance of spliced Flt1. Deletion of the region between AflIII and NheI, has a significant effect on processing to spliced 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 = 4, mean ± SEM, P < 0.001, ANOVA, *P < 0.05 compared to each of the other samples.

Mentions: To determine if the upstream elements could function to switch splicing independently of the distal polyA sites between AfllIII and NheI, we created another series of deletions which did not include the AflIII–NheI region. We demonstrate that the effect of deletions between SmaI and AflIII are small and insignificant compared to the effect of the removal of a AflIII–NheI fragment at the 3′-end of intron 13 confirming that the primary determinant of intronic processing to sFlt1 in this series of constructs is the cis-element containing the distal polyA sites (Figure 4A–C).Figure 4.


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)

The AflIII–NheI region of FLT1 intron 13 is required to significantly reduce splicing of exon 13 to exon 14. (A) Schematic representation of human FLT1 gene in the region of exons 13 and 14. 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 is 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 deletions that do not remove the distal polyA sites do not substantially increase the abundance of spliced Flt1. Deletion of the region between AflIII and NheI, has a significant effect on processing to spliced 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 = 4, mean ± SEM, P < 0.001, ANOVA, *P < 0.05 compared to each of the other samples.
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Figure 4: The AflIII–NheI region of FLT1 intron 13 is required to significantly reduce splicing of exon 13 to exon 14. (A) Schematic representation of human FLT1 gene in the region of exons 13 and 14. 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 is 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 deletions that do not remove the distal polyA sites do not substantially increase the abundance of spliced Flt1. Deletion of the region between AflIII and NheI, has a significant effect on processing to spliced 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 = 4, mean ± SEM, P < 0.001, ANOVA, *P < 0.05 compared to each of the other samples.
Mentions: To determine if the upstream elements could function to switch splicing independently of the distal polyA sites between AfllIII and NheI, we created another series of deletions which did not include the AflIII–NheI region. We demonstrate that the effect of deletions between SmaI and AflIII are small and insignificant compared to the effect of the removal of a AflIII–NheI fragment at the 3′-end of intron 13 confirming that the primary determinant of intronic processing to sFlt1 in this series of constructs is the cis-element containing the distal polyA sites (Figure 4A–C).Figure 4.

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