Dataset Information

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

ABSTRACT:

Background

Connexin55.5 (Cx55.5) is a gap junction protein with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the receptor-horizontal cell complex in form of hemichannels where light-dependent plasticity occurs. This connexin is the first example of a gap junction protein processed to form two protein isoforms from a monocistronic message by an IRES mediated process. The nuclear occurrence of a carboxy-terminal fragment of this protein provides evidence that this gap junction protein may participate in a putative cytoplasmic to nuclear signal transfer.

Results

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.

Conclusion

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.

SUBMITTER: Ul-Hussain M

PROVIDER: S-EPMC2579433 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Publications

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

Ul-Hussain Mahboob M Dermietzel Rolf R Zoidl Georg G

BMC molecular biology 20081023

<h4>Background</h4>Connexin55.5 (Cx55.5) is a gap junction protein with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the receptor-horizontal cell complex in form of hemichannels where light-dependent plasticity occurs. This connexin is the first example of a gap junction protein processed to form two protein isoforms from a monocistronic message by an IRES mediated process. The nuclear occurrence of a carboxy-terminal fragment of this protein provides ...[more]

PMID: 18947383

Similar Datasets

Project description:BACKGROUND: Earlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation (Dhar et al, Nucleic Acids Res, 2007). The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation. METHODOLOGY/PRINCIPAL FINDINGS: We have probed the putative secondary structure of the IRF2 5'UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factor, polypyrimidine tract binding protein (PTB). Deletion of 25 nts from the 3'terminus of the 5'untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2-5'UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3'end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Finally, we have demonstrated that addition of recombinant PTB enhances ribosome assembly on IRF2 IRES suggesting possible role of PTB in mediating internal initiation of translation of IRF2 RNA. CONCLUSION/SIGNIFICANCE: It appears that PTB binding to multiple sites within IRF2 5'UTR leads to a conformational change in the RNA that facilitate binding of other trans-acting factors to mediate internal initiation of translation.

Dataset Information

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

Background

Results

Conclusion

Publications

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

Similar Datasets

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