A New 34-Kilodalton Isoform of Human Fibroblast Growth Factor 2 Is Cap Dependently Synthesized by Using a Non-AUG Start Codon and Behaves as a Survival Factor

Abstract

Four isoforms of human fibroblast growth factor 2 (FGF-2) result from alternative initiations of translation at three CUG start codons and one AUG start codon. Here we characterize a new 34-kDa FGF-2 isoform whose expression is initiated at a fifth initiation codon. This 34-kDa FGF-2 was identified in HeLa cells by using an N-terminal directed antibody. Its initiation codon was identified by site-directed mutagenesis as being a CUG codon located at 86 nucleotides (nt) from the FGF-2 mRNA 5′ end. Both in vitro translation and COS-7 cell transfection using bicistronic RNAs demonstrated that the 34-kDa FGF-2 was exclusively expressed in a cap-dependent manner. This contrasted with the expression of the other FGF-2 isoforms of 18, 22, 22.5, and 24 kDa, which is controlled by an internal ribosome entry site (IRES). Strikingly, expression of the other FGF-2 isoforms became partly cap dependent in vitro in the presence of the 5,823-nt-long 3′ untranslated region of FGF-2 mRNA. Thus, the FGF-2 mRNA can be translated both by cap-dependent and IRES-driven mechanisms, the balance between these two mechanisms modulating the ratio of the different FGF-2 isoforms. The function of the new FGF-2 was also investigated. We found that the 34-kDa FGF-2, in contrast to the other isoforms, permitted NIH 3T3 cell survival in low-serum conditions. A new arginine-rich nuclear localization sequence (NLS) in the N-terminal region of the 34-kDa FGF-2 was characterized and found to be similar to the NLS of human immunodeficiency virus type 1 Rev protein. These data suggest that the function of the 34-kDa FGF-2 is mediated by nuclear targets.

ACKNOWLEDGMENTS

Emmanuelle Arnaud and Christian Touriol contributed equally to this work.

We thank F. Bayard, B. Bugler, and B. Galy for helpful discussions, D. Villa for pictures, D. Warwick for English proofreading, and C. Zanibellato for technical assistance. We thank R. Z. Florkiewicz for the gift of the genomic DNA FGF-2 sequence. We also thank E. Bieth for the poly(A)-containing plasmid and A. Ramackers for plasmid pSCT-DOG.

This work was supported by grants from the Association pour la Recherche sur le Cancer, the Agence Nationale de Recherches sur le SIDA, the Ligue Nationale contre le Cancer, the Conseil Régional Midi-Pyrénées, and the European Community Biotechnology Program (subprogram Cell Factory, Actions de Recherches Concertées, contract 94/99-181). E. Arnaud received successive fellowships from the Association pour la Recherche sur le Cancer and from the Ligue Régionale contre le Cancer. C. Touriol received successive fellowships from the Ministère de l’Education Nationale et de la Recherche and from the Ligue Nationale contre le Cancer.