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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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Identification of sFlt1 mRNA proximal intronic cleavage site with the reporter vector pCβS. (A) Schematic representation of the pCβS vector with sFlt1 proximal fragment inserted downstream of the CMV promoter to create pCβs/sFlt1 prox. (B and C) pCβS/sFlt1 prox or pCβs/sFlt1proxmut was transfected into HTR-8/SVNeo cells and then isolated mRNA was probed by RPA with a cRNA derived from the corresponding plasmid. A strong band in (B) lane 1 at ∼170 represents cleavage directed by the putative proximal intronic sFlt1 poly(A) signal sequence. Mutation of the proximal signal leads to loss of the primary cleavage at ∼170 in (C) lane 1. A weak band at ∼190 seen in lane 1 (C), probably represents cleavage directed by a cryptic poly(A) signal sequence. The signal sequence is boxed in sequence above (B and C). Digested RNA samples were run alongside yeast RNA (negative control) and (undigested) probe.
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Figure 6: Identification of sFlt1 mRNA proximal intronic cleavage site with the reporter vector pCβS. (A) Schematic representation of the pCβS vector with sFlt1 proximal fragment inserted downstream of the CMV promoter to create pCβs/sFlt1 prox. (B and C) pCβS/sFlt1 prox or pCβs/sFlt1proxmut was transfected into HTR-8/SVNeo cells and then isolated mRNA was probed by RPA with a cRNA derived from the corresponding plasmid. A strong band in (B) lane 1 at ∼170 represents cleavage directed by the putative proximal intronic sFlt1 poly(A) signal sequence. Mutation of the proximal signal leads to loss of the primary cleavage at ∼170 in (C) lane 1. A weak band at ∼190 seen in lane 1 (C), probably represents cleavage directed by a cryptic poly(A) signal sequence. The signal sequence is boxed in sequence above (B and C). Digested RNA samples were run alongside yeast RNA (negative control) and (undigested) probe.

Mentions: The results thus far suggested that the region containing the distal polyA signal sequences had a profound effect on polyadenylation of sFlt1. We had also identified, by sequence analysis, a putative proximal polyA signal sequence just upstream of the polyadenylation site of the 2.6 Kb sFlt1 transcript (11). To study the regulation of this short transcript, we first performed a polyadenylation assay to confirm the identity of the proximal poly(A) signal sequence. We cloned a genomic DNA fragment from FLT1 that included the putative proximal mRNA cleavage site into pCβS and introduced this construct into HTR-8/SVNeo, a trophoblast cell line (Figure 6A). Cleavage of the resulting chimeric transcript is directed by a polyA signal sequence within the cloned FLT1 fragment and was identified by an RPA using an antisense cRNA probe derived from the sFlt1 construct. RNA from cells transfected with the proximal sFlt1 construct sequence showed a strong signal at ∼170 nt, which is ∼20 nt downstream of the putative proximal sFlt1 poly(A) signal sequence, where we would predict RNA cleavage and polyadenylation would occur (Figure 6B). When the putative signal sequence ATTAAA was mutated, cleavage of the chimeric transcript was abolished confirming the identity of the proximal intronic sFlt1 polyA signal sequence (Figure 6C). This signal sequence is quite interesting and unusual as it contains the stop codon for intronic sFlt1, thus defining both the COO terminus of intronic sFlt1 and the 3′-end of this transcript.Figure 6.


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)

Identification of sFlt1 mRNA proximal intronic cleavage site with the reporter vector pCβS. (A) Schematic representation of the pCβS vector with sFlt1 proximal fragment inserted downstream of the CMV promoter to create pCβs/sFlt1 prox. (B and C) pCβS/sFlt1 prox or pCβs/sFlt1proxmut was transfected into HTR-8/SVNeo cells and then isolated mRNA was probed by RPA with a cRNA derived from the corresponding plasmid. A strong band in (B) lane 1 at ∼170 represents cleavage directed by the putative proximal intronic sFlt1 poly(A) signal sequence. Mutation of the proximal signal leads to loss of the primary cleavage at ∼170 in (C) lane 1. A weak band at ∼190 seen in lane 1 (C), probably represents cleavage directed by a cryptic poly(A) signal sequence. The signal sequence is boxed in sequence above (B and C). Digested RNA samples were run alongside yeast RNA (negative control) and (undigested) probe.
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Figure 6: Identification of sFlt1 mRNA proximal intronic cleavage site with the reporter vector pCβS. (A) Schematic representation of the pCβS vector with sFlt1 proximal fragment inserted downstream of the CMV promoter to create pCβs/sFlt1 prox. (B and C) pCβS/sFlt1 prox or pCβs/sFlt1proxmut was transfected into HTR-8/SVNeo cells and then isolated mRNA was probed by RPA with a cRNA derived from the corresponding plasmid. A strong band in (B) lane 1 at ∼170 represents cleavage directed by the putative proximal intronic sFlt1 poly(A) signal sequence. Mutation of the proximal signal leads to loss of the primary cleavage at ∼170 in (C) lane 1. A weak band at ∼190 seen in lane 1 (C), probably represents cleavage directed by a cryptic poly(A) signal sequence. The signal sequence is boxed in sequence above (B and C). Digested RNA samples were run alongside yeast RNA (negative control) and (undigested) probe.
Mentions: The results thus far suggested that the region containing the distal polyA signal sequences had a profound effect on polyadenylation of sFlt1. We had also identified, by sequence analysis, a putative proximal polyA signal sequence just upstream of the polyadenylation site of the 2.6 Kb sFlt1 transcript (11). To study the regulation of this short transcript, we first performed a polyadenylation assay to confirm the identity of the proximal poly(A) signal sequence. We cloned a genomic DNA fragment from FLT1 that included the putative proximal mRNA cleavage site into pCβS and introduced this construct into HTR-8/SVNeo, a trophoblast cell line (Figure 6A). Cleavage of the resulting chimeric transcript is directed by a polyA signal sequence within the cloned FLT1 fragment and was identified by an RPA using an antisense cRNA probe derived from the sFlt1 construct. RNA from cells transfected with the proximal sFlt1 construct sequence showed a strong signal at ∼170 nt, which is ∼20 nt downstream of the putative proximal sFlt1 poly(A) signal sequence, where we would predict RNA cleavage and polyadenylation would occur (Figure 6B). When the putative signal sequence ATTAAA was mutated, cleavage of the chimeric transcript was abolished confirming the identity of the proximal intronic sFlt1 polyA signal sequence (Figure 6C). This signal sequence is quite interesting and unusual as it contains the stop codon for intronic sFlt1, thus defining both the COO terminus of intronic sFlt1 and the 3′-end of this transcript.Figure 6.

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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