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

Nucleic Acids Res. 2010 Aug;38(15):5130-40. doi: 10.1093/nar/gkq198. Epub 2010 Apr 12.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alternative Splicing*
  • Animals
  • Base Sequence
  • COS Cells
  • Cells, Cultured
  • Chlorocebus aethiops
  • Conserved Sequence
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / metabolism
  • Humans
  • Introns*
  • Polyadenylation*
  • Regulatory Sequences, Ribonucleic Acid*
  • Trophoblasts / metabolism
  • Vascular Endothelial Growth Factor Receptor-1 / genetics*
  • Vascular Endothelial Growth Factor Receptor-1 / metabolism

Substances

  • Regulatory Sequences, Ribonucleic Acid
  • Vascular Endothelial Growth Factor Receptor-1