New vascular endothelial growth factor isoform generated by internal ribosome entry site-driven CUG translation initiation.

We recently demonstrated that the very long 5'-untranslated region (5'-UTR) of the vascular endothelial growth factor (VEGF) mRNA contains two independent internal ribosome entry sites (IRES A and B). In the human sequence, four potential CUG translation initiation codons are located in between these IRES and are in frame with the classical AUG start codon. By in vitro translation and COS-7 cell transfections, we demonstrate that a high mol wt VEGF isoform [called large VEGF (L-VEGF)] is generated by an alternative translation initiation process, which occurs at the first of these CUG codons. Using a bicistronic strategy, we show that the upstream IRES B controls the translation initiation of L-VEGF. This isoform is 206 amino acids longer than the classical AUG-initiated form. With a specific antibody raised against this NH2 extension, we show that the L-VEGF is present in different mouse tissues or in transfected COS-7 cells. We also demonstrate that L-VEGF is cleaved into two fragments: a 23-kDa NH2-specific fragment and a fragment with an apparent size similar to that of the classical AUG-initiated form. This cleavage requires the integrity of a hydrophobic sequence located in the central part of the L-VEGF molecule. This sequence actually plays the role of signal peptide in the classical AUG-initiated form. The AUG-initiated form and the COOH cleavage product of the L-VEGF are both secreted. In contrast, the large isoform and its NH2 fragment present an intracellular localization. These data unravel a further level of complexity in the regulation of VEGF expression.

FIF [fibroblast growth factor-2 (FGF-2)-interacting-factor], a nuclear putatively antiapoptotic factor, interacts specifically with FGF-2.

Numerous evidence indicates that some of the activities of fibroblast growth factor 2 (FGF-2) depend on an intracrine mode of action. Recently, we showed that three high molecular mass (HMM) nuclear forms of FGF-2 are part of a 320-kDa protein complex while the cytoplasmic AUG-initiated form is included in a 130-kDa complex. Consequently, the characterization of FGF endogenous targets has become crucial to allow the elucidation of their endogenous activities. Through the screening of GAL4-based yeast two-hybrid expression libraries, we have isolated a gene encoding a nuclear protein of 55 kDa, FIF (FGF-2-interacting-factor), which interacts specifically with FGF-2 but not with FGF-1, FGF-3, or FGF-6. In this system, FIF interacts equally well with the NH2-extended 24-kDa FGF form as with the 18-kDa form, indicating that the FIF-binding motif is located in the last 155 amino acids of FGF-2. Nevertheless, coimmunoprecipitation experiments showed an exclusive association with HMM FGF-2. The predicted protein contains a canonical leucine zipper domain and three overlapping hydrophobic heptad repeats. The region spanning these repeats is, together with a region located in the N-terminal part of the FIF protein, implicated in the binding to FGF-2. In contrast to the full-length FIF protein, several deletion constructs were able to transactivate a lac-Z reporter gene. Furthermore, the COOH-terminal part, but not the full-length FIF protein, has previously been shown to exhibit antiapoptotic properties. Thus we discuss the possibility that these activities could reflect a physiological function of FIF through its interaction with FGF-2.

Two independent internal ribosome entry sites are involved in translation initiation of vascular endothelial growth factor mRNA.

The mRNA of vascular endothelial growth factor (VEGF), the major angiogenic growth factor, contains an unusually long (1,038 nucleotides) and structured 5' untranslated region (UTR). According to the classical translation initiation model of ribosome scanning, such a 5' UTR is expected to be a strong translation inhibitor. In vitro and bicistronic strategies were used to show that the VEGF mRNA translation was cap independent and occurred by an internal ribosome entry process. For the first time, we demonstrate that two independent internal ribosome entry sites (IRESs) are present in this 5' UTR. IRES A is located within the 300 nucleotides upstream from the AUG start codon. RNA secondary structure prediction and site-directed mutagenesis allowed the identification of a 49-nucleotide structural domain (D4) essential to IRES A activity. UV cross-linking experiments revealed that IRES A activity was correlated with binding of a 100-kDa protein to the D4 domain. IRES B is located in the first half of the 5' UTR. An element between nucleotides 379 and 483 is required for its activity. Immunoprecipitation experiments demonstrated that a main IRES B-bound protein was the polypyrimidine tract binding protein (PTB), a well-known regulator of picornavirus IRESs. However, we showed that binding of the PTB on IRES B does not seem to be correlated with its activity. Evidence is provided of an original cumulative effect of two IRESs, probably controlled by different factors, to promote an efficient initiation of translation at the same AUG codon.

The Rhizobium meliloti PII protein, which controls bacterial nitrogen metabolism, affects alfalfa nodule development.

Symbiotic nitrogen fixation involves the development of specialized organs called nodules within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. To determine the importance of bacterial nitrogen metabolism in symbiosis, we have characterized a key regulator of this metabolism in Rhizobium meliloti, the uridylylatable P(II) protein encoded by glnB. We have constructed both a glnB null mutant and a point mutant making nonuridylylatable P(II). In free-living conditions, P(II) is required for expression of the ntrC-dependent gene glnII and for adenylylation of glutamine synthetase I. P(II) is also required for efficient infection of alfalfa but not for expression of nitrogenase. However alfalfa plants inoculated with either glnB mutant are nitrogen-starved in the absence of added combined nitrogen. We hypothesize that P(II) controls expression or activity of a bacteroid ammonium transporter required for a functional nitrogen-fixing symbiosis. Therefore, the P(II) protein affects both Rhizobium nitrogen metabolism and alfalfa nodule development.

Symbiotic nitrogen fixation does not require adenylylation of glutamine synthetase I in Rhizobium meliloti.

Symbiotic nitrogen fixation is accompanied by a shift of Rhizobium nitrogen metabolism from ammonium assimilation to ammonium export, which probably involves genetic or metabolic regulation of glutamine synthetase activity. In free-living Rhizobium meliloti glutamine synthetase I (GSI) is regulated post-translationally by reversible adenylylation in response to ammonium addition. Moreover, full expression of the GSI gene glnA requires the transcriptional activator, NtrC. A glnA1 mutant synthesizing a non-adenylylatable GSI produces normal nitrogen-fixing nodules on alfalfa: GSI adenylylation is dispensable for symbiotic nitrogen fixation. This is rationalized by the observation that less GS protein is present in R. meliloti bacteroids than in free-living bacterial cells.