Translation of Vascular Endothelial Growth Factor mRNA by Internal Ribosome Entry: Implications for Translation under Hypoxia

Abstract

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic growth factor that promotes compensatory angiogenesis in circumstances of oxygen shortage. The requirement for translational regulation of VEGF is imposed by the cumbersome structure of the 5 untranslated region (5UTR), which is incompatible with efficient translation by ribosomal scanning, and by the physiologic requirement for maximal VEGF production under conditions of hypoxia, where overall protein synthesis is compromised. Using bicistronic reporter gene constructs, we show that the 1,014-bp 5UTR of VEGF contains a functional internal ribosome entry site (IRES). Efficient cap-independent translation is maintained under hypoxia, thereby securing efficient production of VEGF even under unfavorable stress conditions. To identify sequences within the 5UTR required for maximal IRES activity, deletion mutants were analyzed. Elimination of the majority (851 nucleotides) of internal 5UTR sequences not only maintained full IRES activity but also generated a significantly more potent IRES. Activity of the 163-bp long "improved" IRES element was abrogated, however, following substitution of a few bases near the 5 terminus as well as substitutions close to the translation start codon. Both the full-length 5UTR and its truncated version function as translational enhancers in the context of a monocistronic mRNA.

Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes

“…In addition, while the viral IRES is about 450 bp in length, mammalian IRESes range in size from under 100 bp 14 to approximately 1 kb. 11 For example, the smaller mammalian IRESes from the GTX (glial and testis-specific homeobox protein) or BIP genes may be useful for incorporation into vectors with limited packaging capacity, such as those derived from AAV (adeno-associated virus) or SV40 (simian virus 40).…”

“…12 However, it has been reported that a truncated version of the mouse Vegf IRES containing the 5' end of the Vegf 5'UTR and a portion of IRES A is better than a full-length 1 kb fragment containing both IRES A and IRES B, in mediating downstream gene expression. 11 We therefore compared the performances of the human full-length (VEGF IRES) and truncated (V163 IRES) versions against the EMCV IRES in KB-3-1 cells. The results show the V163 IRES to be capable of mediating significantly better downstream gene expression than either the complete VEGF IRES (P Ͻ 0.05) or EMCV IRES (P Ͻ 0.05) (Figure 4).…”

“…7 IRES sequences that have been identified in mammalian genes include those of the immunoglobulin heavy chain binding protein (BIP), 8 eukaryotic initiation factor 4G (EIF4G), 9 c-myc proto-oncogene (MYC), 10 and vascular endothelial growth factor (VEGF). 11,12 Thus far, they have not been utilized in gene therapy vectors for the coexpression of multiple genes. While most of the IRESes from viral genes are approximately 450 nucleotides in length, IRESes from mammalian genes can range in size from approximately 100 bp to more than 1 kb.…”

“…Unlike viral IRESes, mammalian IRESes often serve as recognition sites for translational regulatory factors, thereby mediating post-transcriptional regulation of gene expression. Examples include IRES-mediated translation of VEGF mRNA, which remains efficient even under unfavorable hypoxic conditions when overall translation is reduced, 11 and stress-modulated translation of FGF-2. 13 The respective IRESes located at the 5'UTR of these genes have been suspected to be responsible for this regulation.…”

A mutation in the c-myc-IRES leads to enhanced internal ribosome entry in multiple myeloma: A novel mechanism of oncogene de-regulation

“…The latter is mediated by structured regions of RNA termed internal ribosome entry segments (IRES), and these have the ability to direct ribosome binding some considerable distance (up to 1000 nts) from the 5' end of the message. IRESs were originally discovered in picornaviral mRNAs, but have now been identi®ed in a number of eukaryotic mRNAs, the protein products of which are involved in the control of cell growth including, FGF-2 (Vagner et al, 1995), VEGF (Miller et al, 1998;Stein et al, 1998), PDGF (Bernstein et al, 1997) and c-myc (Nanbru et al, 1997;Stoneley et al, 1998) and cell death Holcik et al, 1999). In the case of c-myc we have shown that initiation of protein synthesis can occur by two distinct mechanisms; cap-dependent scanning and IRES mediated .…”