A Single Internal Ribosome Entry Site Containing a G Quartet RNA Structure Drives Fibroblast Growth Factor 2 Gene Expression at Four Alternative Translation Initiation Codons

Abstract

The 484-nucleotide (nt) alternatively translated region (ATR) of the human fibroblast growth factor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. Although the 5-end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis-acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5-ATR led us to define a 176-nt region as being necessary and sufficient for IRES function at four codons present in a downstream 308-nt RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5-ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G quartet motif that constitute novel structural determinants of IRES function. The human FGF-21 gene belongs to a gene family of 23 fibroblast growth factors and is involved in various fundamental cellular processes, such as cell proliferation, differentiation, and angiogenesis (1). The contribution of translational control in FGF-2 gene expression has been particularly well documented. First, a process of alternative initiation of translation occurs on the unique FGF-2 mRNA and leads to the production of five FGF-2 protein isoforms with extended NH 2 -terminal ends (2-4). Second, translation of four of these isoforms is initiated at non-canonical CUG codons (2-4). Third, the use of alternative initiation codons is controlled in transformed and stressed cells (5). Fourth, protein synthesis on the FGF-2 mRNA can occur by internal ribosome entry (6).Whereas the usual mechanism of translation in eukaryotes involves the recruitment of the 40 S ribosomal subunit to the 5Ј-cap structure of the mRNA, a restricted but growing number of viral and cellular mRNA initiate their translation through the recruitment of the 40 S ribosomal subunit to internal sequences of the mRNA called IRES (7-9). As regards the FGF-2 mRNA, this process is active in vivo in transgenic mice (10). Interestingly, the FGF-2 IRES is able to contribute to the choice of initiation codons because translation at the cap-proximal CUG codon occurs by a cap-dependent process, whereas translation at the other four codons occurs by an IRES-based mechanism (4). To understand translation initiation codon selection in the FGF-2 mRNA, it is therefore important to define the cis-elements required for FGF-2 IRES function.The RNA sequences and structural features of cellular IRESs remain largely unknown. Various attempts to define the cis-acting elements required for the function of cellular IRES have failed to find a common RNA sequence. Cellular IRES seem to be very diverse in nature, without stringent sequence similarity. Because IRES elements in viral RNA genomes contain higher order structures whose inte...

Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression

“…The situation is further complicated by the observation that the 9 nt Gtx element can function as a translation repressor when present in the 5 0 UTR of a monocistronic mRNA (Hu et al, 1999). Furthermore, an identical sequence also appears in the fgf2 IRES and in this case appears to have no effect on internal initiation (Bonnal et al, 2003). Thus the evidence to date suggests that cellular IRESs differ to some extent from their viral counterparts.…”

“…Primary sequence elements that are implicated in internal initiation have been identified in the IRESs of Gtx, Rbm3 and fgf2 Bonnal et al, 2003;Chappell and Mauro, 2003). Short-sequence modules found in the Gtx and Rbm3 IRESs can promote internal initiation and have been shown to bind to the 40S ribosome in cell-free assays.…”

“…However, thus far there is no obvious conservation of secondary structure between these IRESs. Currently structural models have been determined using chemical and enzymatic probing for only a handful of cellular IRESs (LeQuesne et al, 2001;Bonnal et al, 2003;Jopling et al, 2004;Mitchell et al, 2003;Yaman et al, 2003). To date no common structural features have been found, even when the IRESs of the closely related genes c-myc and L-myc are compared (Figure 2) (LeQuesne et al, 2001;Jopling et al, 2004).…”

Selective translation of mRNAs in the left ventricular myocardium of the mouse in response to acute pressure overload

“…Although the precise effects of eIF4E phosphorylation on translational efficiency of specific mRNAs have not been elucidated in cardiac muscle, we have shown in previous studies that eIF4E phosphorylation is increased in response to pressure overload hypertrophy [7,29]. Further evidence indicates that the mRNAs coding for MDM2, c-myc, c-jun, and FGF-2 contain Internal Ribosome Entry Sites (IRES), which are elements in the 5′-UTR that direct translational initiation independent of the 7 mGppp cap [30][31][32][33]. The presence of an IRES could target specific mRNAs for translation in response to a growth stimulus b facilitating capindependent initiation However, the physiological relevance of IRES elements as a mechanism of translational initiation is controversial [34].…”

Evolutionary patterns of non-coding RNAs

“…For example, the human fibroblast growth factor 2 contains 5 translation initiation codons. Translation initiation of the codon proximal to the 5'-end is initiated by a cap-dependent process, whereas initiation of the remaining codons depends on the IRES [33]. IRES elements are defined by functional criteria and cannot yet be predicted by the presence of characteristic RNA sequence or structural motifs.…”