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This Article Full Text Full Text (PDF) All Versions of this Article: fj.07-8809comv1 21/14/3885    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Thomas, C. P. Articles by Liu, K. Z. PubMed PubMed Citation Articles by Thomas, C. P. Articles by Liu, K. Z.

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

Christie P. Thomas^*,,1, Janet I. Andrews and Kang Z. Liu^*

Departments of
^* Internal Medicine and

Obstetrics and Gynecology, University of Iowa and the

Veterans Affairs Medical Center, Iowa City, Iowa, USA

¹Correspondence: Department of Internal Medicine and Graduate Program in Molecular and Cellular Biology, E300 GH, University of Iowa Carver College of Medicine, 200 Hawkins Dr., Iowa City, IA 52242-1081 USA. E-mail: christie-thomas{at}uiowa.edu

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 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 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.—Thomas, C. P., Andrews, J. I., Liu, K. Z. Intronic polyadenylation signal sequences and alternate splicing generate human soluble Flt1 variants and regulate the abundance of soluble Flt1 in the placenta.

Key Words: angiogenesis • soluble VEGF receptor • preeclampsia