Intronic polyadenylation signal sequences and alternate splicing generate human soluble Fltl variants and regulate the abundance of soluble Flt1 in the placenta

Abstract

The gene FLT1 produces at least two transcripts from a common transcription start site: full-length Flt1 contains 30 exons encoding a membrane-bound VEGF receptor; soluble Flt1 (sFlt1) shares the first 13 exons but utilizes poly(A) signal sequences within intron 13 to create a transcript that lacks downstream exons. To address the mechanisms that regulate human sFlt1, we mapped the 3' end of sFlt1 mRNA and defined the full extent of its 3' untranslated region (UTR). We identified a 3.2 Kb sFlt1 transcript that is cleaved within an alternatively spliced exon downstream of exon 14 and is predicted to encode a C-terminal variant of sFlt1 with an unusual polyserine tail. sFlt1 mRNA cleavage sites within intron 13 were identified in human placenta and in vascular endothelium by ribonuclease protection assay (RPA). A proximal and two distal mRNA cleavage sites were identified by RPA downstream of consensus polyadenylation signals that create variant transcripts with a 3' UTR ranging from 30 bases to approximately 4 Kb. Northern blot analysis and 3' rapid amplification of cDNA ends (RACE) in placenta confirmed the existence of distal intronic sFlt1 cleavage sites that give rise to a sFlt1 transcript of approximately 7 Kb. The identity of the distal signal sequences were then confirmed by mutagenesis of putative signal elements in a polyadenylation reporter assay. We demonstrate the heterogeneity of human sFlt1 that arises from alternate splicing and from alternative polyadenylation directed by strong intronic poly(A) signal sequences leading to C-terminal variants and to an sFlt1 transcript with a large 3' UTR containing several AU rich elements and poly(U) regions that may regulate mRNA stability.

Prevention of gravidic endothelial hypertension by aspirin treatment administered from the 8th week of gestation

“…[10][11][12] These soluble molecules may be generated by changes in the splicing of mRNA, such as the e15 isoform of sFLT1, [13][14][15] and are thought to have the role of 'molecular traps' for their active ligands.…”

A Maternally Sequestered, Biopolymer‐Stabilized Vascular Endothelial Growth Factor (VEGF) Chimera for Treatment of Preeclampsia

“…VEGF‐A binds to the VEGFR2 receptor, and by protein kinase C (PKC)–MEK signaling, acts on the VEGF‐A Response Element on Intron 13 of the VEGFR1 gene 94. Premature polyadenylation sites are located near the VEGF‐A Response Element on Intron 13, which then trigger alternative splicing of the VEGFR1 gene into sFlt‐1 mRNA transcripts 96, 97, 98. In this study, all rats that received ELP‐VEGF at a dose of 5 mg/kg per day had increased total plasma sFlt‐1 levels (Figure 4A and B).…”

Jumonji domain-containing protein 6 (Jmjd6) is required for angiogenic sprouting and regulates splicing of VEGF-receptor 1

“…Jmjd6 silencing augmented the expression of the alternative splice variant of Flt1 that contains exons 1 to 13 of Flt1, leading to the generation of a protein that lacks the transmembrane and intracellular kinase domain, hence functioning as a soluble VEGF-and PlGF-trapping molecule. Soluble Flt1 was initially cloned in 1993 (24) and, since then, additional splice variants that terminate with exon 14 and exon 15a or 15b have been identified, some of which are specifically expressed in smooth-muscle cells (25)(26)(27). Although the detailed biological and molecular functions remain to be determined, increasing evidence suggest that sFlt1 is up-regulated by hypoxia in the placenta and plays a causal role in the pathogenesis of preeclampsia (28,29).…”