Poly(A)-binding protein-interacting protein 2, a strong regulator of vascular endothelial growth factor mRNA.
Expression of vascular endothelial growth factor (VEGF) is tightly regulated, particularly at the level of its mRNA stability, which is essentially mediated through the 3'-untranslated region (3'-UTR) of VEGF mRNA. To identify new protein partners regulating VEGF mRNA stability, we screened a cDNA expression library with an RNA probe corresponding to the entire VEGF mRNA 3'-UTR. We identified the "poly(A)-binding protein-interacting protein 2" (PAIP2) as a new VEGF mRNA 3'-UTR interacting protein. By RNA electromobility shift assays, we showed that PAIP2 binds to two distinct regions of a domain encompassing base 1 to 1280 of the VEGF 3'-UTR. Such in vitro interaction was confirmed using cell extracts in which PAIP2 expression is induced by tetracycline (Tet-on cells). Moreover, we demonstrated by RNA affinity purification as well as by ribonucleoprotein complexes immunoprecipitation, that PAIP2 interacts with VEGF mRNA in vivo. Using an in vitro RNA degradation assay, the half-life of VEGF 3'-UTR was found to be increased by overexpressing PAIP2. PAIP2 stabilizes endogenous VEGF mRNA in Tet-on cells, leading to increased VEGF secretion. Moreover, RNAi-mediated knock-down of PAIP2 significantly reduces the steady-state levels of endogenous VEGF mRNA. We also showed, by in vitro protein-protein interactions and co-immunoprecipitation experiments, that PAIP2 interacts with HuR, an already known VEGF mRNA-binding protein, suggesting cooperation of both proteins for VEGF mRNA stabilization. Hence, PAIP2 appears to be a crucial regulator of VEGF mRNA and as a consequence, any variation in its expression could modulate angiogenesis.
Onesto
Cercina
C
Institute of Signaling, Developmental Biology and Cancer Research, CNRS-UMR 6543, Centre Antoine Lacassagne, 33 avenue de Valombrose, 06189 Nice cedex 2, France.
Berra
Edurne
E
Grépin
Renaud
R
Pagès
Gilles
G
D020413:3' Untranslated Regions; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D000860:Hypoxia; D051379:Mice; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors; D014018:Tissue Distribution; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D020413:3' Untranslated Regions; D002352:Carrier Proteins; D018076:DNA, Complementary; C488448:PAIP2 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D042461:Vascular Endothelial Growth Factor A; D012313:RNA; D005982:Glutathione Transferase; D013752:Tetracycline