Characterization of the internal IRES element of the zebrafish connexin55.5 reveals functional implication of the polypyrimidine tract binding protein

We characterized the IRES element of Cx55.5 in terms of sequence elements necessary for its activity and protein factor(s), which may play a role for its function. Two stretches of polypyrimidine tracts designated PPT1 and PPT2 which influence the IRES activity of this neuronal gap junction protein were identified. Selective deletion of PPT1 results in an appreciable decrease of the IRES activity, while the deletion of PPT2 results in a complete loss. RNA-EMSA and UV-cross linking experiments showed that protein complexes bind to this IRES element, of which the polypyrimidine tract binding protein (PTB) was identified as one of the interacting partners with influence on IRES activity. These results indicate that PTB conveys a role in the regulation of the IRES activity of Cx55.5.Our findings indicate that the activity of the IRES element of the neuronal gap junction protein Cx55.5 is subject of regulation through flanking polypyrimidine tracts, and that the non-canonical trans-activation factor PTB plays an essential role in this process. This observation is of considerable importance and may provide initial insight into molecular-functional relationships of electrical coupling in horizontal cells.Cap-dependent translation is not the only means by which mRNA translation can be initiated. The discovery of internal ribosome entry sites (IRES) in picornaviruses mRNA revealed that the small ribosomal subunit could bind within the mRNA in a cap-independent manner [1,2]. Because of this property, IRES elements provide an exception of the general mechanism of scanning from the 5' end of the cap structure to initiate eukaryotic translation. Multiple IRESs have subsequently been found on different viral mRNAs, and more recently in cellular mRNAs [3-10]. The presence of IRES elements in viruses provides them with the advantage to hijack the translational machinery of the host cell to favor the expression of foreign transcripts. Most of the cellular IRESs have been shown to fu