BACKGROUND
cap-dependent translation is not the only means by which mrna translation can be initiated.
the discovery of internal ribosome entry sites  in picornaviruses mrna revealed that the small ribosomal subunit could bind within the mrna in a cap-independent manner  <cit> .
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  <cit> .
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 function preferentially when cap-dependent translation is physiologically impaired.
consistent with this concept, ires elements were active during γ-radiations  <cit> , hypoxia  <cit>  or amino acid starvation  <cit> .
this led to the current hypothesis that ires-mediated translation of certain mrnas represents a regulatory mechanism that helps the cell to cope with transient stress.
connexins form gap junctions, which are believed to convey a broad spectrum of functions, including the regulation of cell growth, cell differentiation and maintenance of tissue homeostasis  <cit> .
translational initiation of connexin genes has been regarded mainly in a cap-dependent manner, but recent reports have shown that connexins possess ires elements  <cit> , where in one case a single point mutation in an ires element of the 5' untranslated region has been linked to charcot-marie-tooth disease  <cit> .
a recent report on connexin <dig> has shown that this gene use alternate splicing mechanism, which yields transcripts with different 5'-utrs displaying different translational efficiencies  <cit> .
moreover, we reported on the presence of a unique internal ires element in the coding region of the horizontal cell specific zebrafish cx <dig> , which results in an internal translation of a carboxy-terminal domain   <cit> .
the presence of such ires elements in the coding region of eukaryotic genes has only recently become apparent  <cit> .
regulation of a typical caped eukaryotic mrna by modulating the activity of critical translational initiation factors, eif4e and eif4f, is well known  <cit> .
however, the translational regulation of the ires containing mrna is still less understood.
to get insight into the regulation of the ires elements in cellular genes, it becomes imperative to identify the motifs within the ires's scaffold, which are important for its function.
in addition to the requirement of the canonical initiation factors, the requirements of some non-canonical trans-acting factors have been found important for the function of ires elements  <cit> .
in the present study, we extent a previous report on the presence of an internal ires element in the coding region of cx <dig>  by characterizing the ires site in terms of sequence elements important for ires activity and putative trans-acting factors that could modulate the ires function.
our findings indicate that the activity of the ires element is subject of regulation through flanking polypyrimidine tracts, and that ptb seems to be an essential rna binding factor involved in this process.
this observation is of considerable importance since it adds a further facet to the widespread function of ptb.
on one hand ptb has been characterized as a crucial factor involved in the non-canonical trans-activity mechanisms.
there, ptb was initially discovered as a splicing factor due to its ability to bind to polypyrimidine tracts at 3' splice sites  <cit> .
here, our finding supports the previously described role of ptb in ires mediated translation  <cit> , which in our case consists of an internal segment that allows alternate translation of a carboxy-terminal isoform with the capability to translocate to the nucleus.
this finding may provide initial insight on molecular-functional relationships of electrical coupling in horizontal cells, which show a unique light dependent plasticity.
changes of the interneuronal coupling mediated by electrical synapse proteins in response to light adaptation, receptive field shaping and structural plasticity are known as a paramount feature of the outer retina  <cit> .
the regulation of these processes is not understood at the molecular level but information transfer to the nucleus by a locally generated messenger deriving from an electrical synapse protein may comprise a feasible molecular determinant in a yet uncharacterized signalling pathway.
on the basis of our results it is tempting to speculate whether the cx <dig>  ct domain shows a light stimulus dependent translocation and exert its function by interaction with dna, rna and/or other nuclear proteins thus modulating gene regulation making plasticity of horizontal cells possible.
RESULTS
cx <dig>  internal ires element activity is modulated by two polypyrimidine tracts
we reported recently that the carboxy-terminal domain  of the zebrafish cx <dig>  can be internally translated from an ires element present in the coding region of the full length cx <dig>  mrna  <cit> .
a detailed sequence analysis of this element revealed the presence of two stretches of polypyrimidine tracts named polypyrimidine tract  <dig>  and polypyrimidine tract  <dig>  .
we hypothesized that both sequence elements could contribute to ires activity and generated plasmid constructs with deletions of ppt <dig> or ppt <dig> separately or in combination with the ires element  containing the di-cistronic vector prf-ir.
the control di-cis vector prf along with wild type ires di-cis vector prf-ir and the various deletion mutant constructs of the ires element, prf-irdel <dig>  prf-irdel <dig> and prf-irdel <dig> , were transiently transfected into n2a cells and the rennila and firefly luciferase activity measured  <dig> hrs post transfection.
the ires activity was calculated as the ratio of firefly luciferase to rennila luciferase activity .
luciferase activity readings demonstrated that the wild type ires vector  was able to enhance the expression of the downstream located firefly luciferase cistron by ~ <dig> fold as compared to the control vector prf .
deletion of the ppt <dig> element in the plasmid prf-irdel <dig> reduced luciferase activity by ~ <dig> fold.
the deletion of ppt <dig> sequence in plasmid prf-irdel <dig> alone, or the simultaneous deletion of ppt <dig>  ppt <dig> and the intervening sequence in prf-irdel <dig> reduced the luciferase activity to background.
this effect was further confirmed by western blotting using deletion constructs.
in all constructs, the firefly luciferase gene was replaced by the egfp coding region.
the wild type ires construct pre-ir and the deletion constructs, pre-irdel <dig>  pre-irdel <dig> and pre-irdel <dig>  along with the control vector pre were transiently transfected into the n2a cells .
fourty eight hours post transfection cytosolic cell extracts were prepared and  <dig> μg of the total protein was separated by 10% sds-page.
immunodetection with the anti-gfp antibody showed an enhanced expression of egfp in the construct pre-ir with the wild type ires element  as compared to the control vector pre.
the deletion of the ppt <dig> motif induced a moderate decrease in the expression of egfp as compared to pre-ir.
the deletion of the ppt <dig> motif alone or a deletion of both ppt <dig> and ppt <dig> reduced egfp expression below the detection limit.
this finding confirmed the profound effect of ppt <dig> on ires activity consistent with the result obtained by the reporter assay described above.
polypyrimidine tract binding protein  plays an essential role in the ires activity mediated by ppt <dig> and ppt2
the polypyrimidine tract binding protein  is known to bind to polypyrimidine tracts  <cit> .
we assumed a role of ptb in binding to the two polypyrimidine tracts and thereby modulating ires activity.
this hypothesis was tested by overexpression of the human ptb  in n2a cells together with the wild type ires or ppt deletion constructs .
after transient transfection into n2a cells, rennila and firefly luciferase activity was measured and ratios calculated.
luciferase readings showed that co-transfection of the vector pc1-ptb encoding the exogenously expressed huptb with the wild-type ires construct prf-ir stimulated luciferase activity ~ <dig> fold as compared to control vector prf.
similarly, co-transfection of the ppt <dig> deletion construct, prf-irdel <dig> and pc1-ptb, showed a ~ <dig> fold increase in the luciferase activity.
in contrast, simultaneous expression of the ppt <dig> deletion constructs prf-irdel <dig> and prf-irdel <dig> with huptb did not affect luciferase activity .
these results demonstrate that ppt <dig> is crucial for ptb mediated ires activity, while ppt <dig> seems to play a subsidiary role.
a direct correlation between the expression of ptb and the activity of the ires element was substantiated by western blots using the egfp containing di-cis constructs.
the wild type ires construct pre-ir and the deletion constructs were transiently co-transfected with the pc1-ptb vector into n2a cells .
cell extracts were prepared  <dig> hrs after transfection and  <dig> μg of the total protein was separated on 10% sds gel.
immunodetection with anti-gfp and anti-ptb antibodies showed that the huptb over-expression along with the wild type ires element resulted in an enhanced expression of egfp as compared to endogenous expression of ptb.
co-expression of huptb and the ppt <dig> deletion construct promoted an increased egfp expression, which appeared only slightly reduced when compared to the wild type ires element.
the deletion of the ppt <dig> motif, however, led to a profound reduction of egfp protein expression.
the double mutant lacking ppt <dig> and ppt <dig> was indistinguishable from the control vector pre demonstrating that both ppt motifs are necessary to mediate the full ptb effect, but with different efficiency.
polypyrimidine tract i  and polypyrimidine tract ii  are important for the ires mediated expression of the p11-ct of cx <dig>
the importance of ptb and polypyrimidine tracts for the expression of the ires-dependent translation of the carboxy-terminal fragment  of cx <dig>  was also analyzed using the mutant deficient for both polypyrimidine tract binding sites .
the mutated construct and the wild type cx <dig> -egfp  construct were transiently transfected into n2a cells either in presence or absence of over-expressed huptb.
<dig> hrs post-transfection cytosolic cell extracts were prepared and  <dig> μg of the total protein was separated on 10% sds gels.
immunodetection with anti-gfp showed that the deletion of the ppt <dig> and ppt <dig> motif reduced the expression of p11-ct as compared to the wild type construct .
furthermore, over-expression of huptb with the wild type cx <dig>  construct resulted in the expected enhanced expression of p11-ct, while the over-expression of huptb with the pptdel <dig> mutant promoted p11-ct expression to levels below that of the wild type construct.
a specific ribonucleic-protein complex  assembles on the cx <dig>  ires element
we used the rna electromobility shift assay  to determine whether cellular proteins recognize the cx <dig>  ires element.
for this purpose, a radiolabeled rna probe was incubated with a s <dig> cytosolic n2a protein extract.
as shown in fig.
5a the cytosolic s <dig> extract retarded the migration of the rna probe, leading to the formation of a single dominant rna-protein complex when compared to the control reaction without protein.
formation of this complex was effectively competed by the inclusion of a 50-fold molar excess of homologous unlabeled competitor rna.
additionally, uv cross-linking experiments were performed to further characterize the rna-protein complexes that assemble at the ires element .
when the wild type ires probe, or its deletion mutants were incubated with the s <dig> cytosolic n2a protein extract and uv crosslinked several distinct rna-protein complexes were detected after sds-page.
the crosslinked complexes had apparent molecular masses in the range from  <dig> to  <dig> kda.
a prominent band showed an apparent molecular weight of ~ <dig> kda .
the formation of the cross-linked rna-protein complexes was prevented by the inclusion of a  <dig> fold molar excess of unlabeled homologous competitor rna.
when the deletion mutants pptdel <dig>  pptdel <dig> and pptdel <dig> were applied a reduction of the protein complex at around ~ <dig> kda became apparent, indicating that the deletion of the polypyrimidine tracts weakens the binding of a major rna binding protein.
purified gst-ptb fusion protein is able to bind the ires element
the prominent rna-protein complex of ~ <dig> kda suggested that ptb with an apparent molecular weight of  <dig> kda constitutes the protein binding to the ires element.
we evaluated this assumption with a recombinant gst-ptb fusion protein, which was purified to homogeneity and subjected to rna-emsa.
the wild-type ires rna probe and the deletion mutants were incubated with gst alone or with the gst-ptb fusion protein.
as shown in , gst-ptb was able to retard the migration of the rna probe while gst alone did not show any effect.
the formation of the gst-ptb rna complex was prevented by inclusion of  <dig> fold molar excess of homologous unlabeled competitor rna.
the rna-emsa data were further confirmed by uv cross-linking of the gst-ptb fusion protein to the wild type ires rna, which resulted in the formation of a single rna-protein complex of ~ <dig> kda .
this complex was effectively competed by adding  <dig> fold molar excess of unlabeled homologous rna.
no rna-protein complex was formed by uv cross-linking gst alone to the ires element, which excludes that the gst protein itself has rna binding properties.
the pptdel <dig> ires rna showed a reduced binding when compared to the wild type ires rna.
this effect was even more pronounced when the pptdel <dig> and pptdel <dig> mutants were subjected to cross-linking .
ptb interacts with zfcx <dig>  mrna in vivo under physiological conditions
to prove that ptb interacts with the cx <dig>  mrna in living cells under physiological conditions, ribonucleoprotein immunoprecipitation  assays were performed.
n2a cells were transiently transfected with a cx <dig>  expression vector.
<dig> hrs post transfection cells were harvested in pbs and incubated with formaldehyde to form stable rna-protein complexes.
cells were lysed and ptb was immunoprecipitated from cell lysates using ptb specific antibodies.
the rna was extracted, reverse transcribed to generate cdna and subjected to polymerase chain reaction  with primers specific to the cx <dig>  coding region.
the specificity of this approach was proven using β-actin antibody coated beads and a void control with beads only.
as shown , a specific dna band of about  <dig> bp corresponding to the full length cx <dig>  coding region was pcr amplified using the cdna obtained from the immunoprecipitated rna.
to further confirm the specificity of the immunoprecipated rna, nested primers specific for the carboxy-terminal domain of cx <dig>  were employed that led to the amplification of the expected carboxy-terminal fragment of ~ <dig> bp .
no amplicons were found in controls using the β-actin loaded and void beads.
thus, the rnp-ip assay provides additional evidence that ptb is able to interact with the cx <dig>  mrna in living cells under physiological conditions and strengthens our assumption that ptb constitutes a crucial factor involved in activating ires mediated translation of this neuronal connexin.
discussion and
CONCLUSIONS
internal ribosome entry sites  are rna complexes with extensive secondary structures.
several conserved motifs have been described to be essential in the regulation of ires activity among which polypyrimidine tracts are well documented  <cit> .
polypyrimidine tracts are involved in the regulation of mrna translational mechanisms and the importance of oligopyrimidine sequences have already been reported upstream of aug initiation codons in picornaviruses  <cit>  and in the hepatitis c virus  <cit> .
in continuation of previous studies, we analyzed the internal ires element of cx <dig>  for sequence motifs of trans-acting factor, which are important for the function of ires activities.
sequence analyses showed the presence of two stretches of polypyrimidine tracts, ppt <dig> and ppt <dig> flanking the in frame ires sequence of cx <dig> .
to investigate the influence of these polypyrimidine tracts on the activity of the ires element, we deleted ppt <dig> and ppt <dig> either alone or in combination.
subsequent reporter assays and western blot analyses showed that deletion of ppt <dig> had an appreciable effect on the ires activity, while deletion of ppt <dig> results in complete loss of ires activity.
this result indicates that the  <dig> bp stretch of ppt <dig> serves an important function in determining ires activity of cx <dig>  albeit by altering the rna fold.
in addition to their requirements for eukaryotic initiation factors, the efficiency of most ires elements is augmented by non-canonical initiation factors know as itafs   <cit> .
since our initial results indicated an involvement of polypyrimidine tracts in defining the ires activity of cx <dig> , we investigated the role of the polypyrimidine tract binding  protein.
the ires activity of the wild type ires element and its deletion mutants was analyzed in the presence of endogenous levels of ptb or upon overexpression of the protein by transfection with huptb.
overexpression resulted in a significant increase of ires activity of the wild type ires element.
deletion of the ppt <dig> element led to a significant reduction as compared to the wildtype, but the ires element was still responsive in particular when exogenous huptb was added.
deletion of ppt <dig> alone or in combination with ppt <dig> revealed a complete loss of responsiveness.
our results suggest that the polypyrimidine tract  <dig>  is crucial for the ires activity and that ptb conveys a role in the regulation of the ires activity of cx <dig> .
a plausible explanation for the critical role of ppt <dig> in the ires activity is that it binds to additional crucial trans-acting factor, which are important for the recruitment of the ribosomal translational machinery.
alternatively, this tract is important for the specific rna secondary structure and the role of ptb seems to stabilize the active confirmation of the ires element through binding to the rna scaffold  <cit> .
interestingly, the secondary structure prediction using the rna folding algorithm mfold  <cit>  of the wild type ires element and its deletion mutants  revealed that the wild type ires of cx <dig>  has an extended stem-loop structure with semi-conserved y-like configuration, described also for ires elements found in some picornaviruses  <cit> .
deletion mutation of ppt <dig> showed overall a similar structure when compared to the wild type rna fold with a minor loss of the small stem loop.
in contrast, the deletion mutant ppt <dig> showed a complete remodeling of the structure, which was predicted to be energetically less stable as compared to the wild type ires rna fold.
the ptb protein is very well known in regulating ires activity of both viral ires and cellular ires elements  <cit> .
these data are of particular interest keeping in mind that ptb is primarily a nuclear protein, where it plays a role in regulation of splicing events of eukaryotic mrnas  <cit> .
to become functional in ires related activities, ptb needs to shuttle from the cell nucleus to the cytoplasm.
recent evidence on the involvement of ires elements in internal translation of biologically active protein domains makes it essential that this processes need to be regulated  <cit> .
shuttling events of ptb from the cell nucleus to the cytoplasm can be one step where regulation by internal and external factors becomes effective.
one such mechanism has been described recently  <cit> .
in this study, protein kinase a phosphorylation  was found to modulate shuttling of ptb by phosphorylation of a particular serine residue.
this modification results in an increase of the cytoplasm-directed transport of ptb from the nucleus and couples the pka pathway with translocation events of ptb.
in the context with our recent findings, which demonstrated expression of an ires driven carboxy-terminal fragment  of cx <dig>  and its translocation to the nucleus of horizontal cells a link between ptb shuttling and regulated expression of this fragment is challenging.
<cit> .
due to the prominent role of gap junction coupling in horizontal cells in primary processing of visual stimuli and its dependence on dopamine regulation  <cit> , it will be of considerable interest to examine whether a camp dependent mechanism involving dopamine affects ptb shuttling and contributes to the strength of intercellular horizontal cell coupling.
alternatively, retinoic acid, which exerts a profound effect on synaptic plasticity of horizontal cells and involves pka phosphorylation to execute its signal transmission potency  <cit>  may comprise another candidate, which could operate through the indicated mechanism.
aside of the identification of ptb as a protein binding to the cx <dig>  ires element further binding proteins covering the range of ~ <dig> to  <dig> kda were identified by uv crosslinking experiments.
some of them are in the size range of proteins, which have been described to bind to other ires elements  <cit> .
it remains to be established whether these proteins constitute further component of this complex, and whether they are also involved in functional regulation of the cx <dig>  ires element.
in summary, we demonstrate that the activity of the internal ires element of the horizontal cell specific gap junction protein cx <dig>  is modulated by two polypyrimidine tracts.
we further provide evidence that the polypyrimidine binding protein  works as a non-canonical factor in the regulation of the ires activity.
the physiological meaning of the cx <dig>  ires activity and in consequence the generation of the p11-ct protein in horizontal cell function awaits further clarification.

 cap-dependent translation is not the only means by which mrna translation can be initiated. the discovery of internal ribosome entry sites in picornaviruses mrna revealed that the small ribosomal subunit could bind within the mrna in a cap-independent manner. 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. 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 function preferentially when cap-dependent translation is physiologically impaired. consistent with this concept, ires elements were active during γ-radiations, hypoxia or amino acid starvation. this led to the current hypothesis that ires-mediated translation of certain mrnas represents a regulatory mechanism that helps the cell to cope with transient stress. connexins form gap junctions, which are believed to convey a broad spectrum of functions, including the regulation of cell growth, cell differentiation and maintenance of tissue homeostasis. translational initiation of connexin genes has been regarded mainly in a cap-dependent manner, but recent reports have shown that connexins possess ires elements, where in one case a single point mutation in an ires element of the 5' untranslated region has been linked to charcot-marie-tooth disease. a recent report on connexin has shown that this gene use alternate splicing mechanism, which yields transcripts with different 5'-utrs displaying different translational efficiencies. moreover, we reported on the presence of a unique internal ires element in the coding region of the horizontal cell specific zebrafish cx, which results in an internal translation of a carboxy-terminal domain. the presence of such ires elements in the coding region of eukaryotic genes has only recently become apparent. regulation of a typical caped eukaryotic mrna by modulating the activity of critical translational initiation factors, eif4e and eif4f, is well known. however, the translational regulation of the ires containing mrna is still less understood. to get insight into the regulation of the ires elements in cellular genes, it becomes imperative to identify the motifs within the ires's scaffold, which are important for its function. in addition to the requirement of the canonical initiation factors, the requirements of some non-canonical trans-acting factors have been found important for the function of ires elements. in the present study, we extent a previous report on the presence of an internal ires element in the coding region of cx by characterizing the ires site in terms of sequence elements important for ires activity and putative trans-acting factors that could modulate the ires function. our findings indicate that the activity of the ires element is subject of regulation through flanking polypyrimidine tracts, and that ptb seems to be an essential rna binding factor involved in this process. this observation is of considerable importance since it adds a further facet to the widespread function of ptb. on one hand ptb has been characterized as a crucial factor involved in the non-canonical trans-activity mechanisms. there, ptb was initially discovered as a splicing factor due to its ability to bind to polypyrimidine tracts at 3' splice sites. here, our finding supports the previously described role of ptb in ires mediated translation, which in our case consists of an internal segment that allows alternate translation of a carboxy-terminal isoform with the capability to translocate to the nucleus. this finding may provide initial insight on molecular-functional relationships of electrical coupling in horizontal cells, which show a unique light dependent plasticity. changes of the interneuronal coupling mediated by electrical synapse proteins in response to light adaptation, receptive field shaping and structural plasticity are known as a paramount feature of the outer retina. the regulation of these processes is not understood at the molecular level but information transfer to the nucleus by a locally generated messenger deriving from an electrical synapse protein may comprise a feasible molecular determinant in a yet uncharacterized signalling pathway. on the basis of our results it is tempting to speculate whether the cx ct domain shows a light stimulus dependent translocation and exert its function by interaction with dna, rna and/or other nuclear proteins thus modulating gene regulation making plasticity of horizontal cells possible.  cx internal ires element activity is modulated by two polypyrimidine tracts we reported recently that the carboxy-terminal domain of the zebrafish cx can be internally translated from an ires element present in the coding region of the full length cx mrna. a detailed sequence analysis of this element revealed the presence of two stretches of polypyrimidine tracts named polypyrimidine tract and polypyrimidine tract. we hypothesized that both sequence elements could contribute to ires activity and generated plasmid constructs with deletions of ppt or ppt separately or in combination with the ires element containing the di-cistronic vector prf-ir. the control di-cis vector prf along with wild type ires di-cis vector prf-ir and the various deletion mutant constructs of the ires element, prf-irdel prf-irdel and prf-irdel, were transiently transfected into n2a cells and the rennila and firefly luciferase activity measured hrs post transfection. the ires activity was calculated as the ratio of firefly luciferase to rennila luciferase activity. luciferase activity readings demonstrated that the wild type ires vector was able to enhance the expression of the downstream located firefly luciferase cistron by ~ fold as compared to the control vector prf. deletion of the ppt element in the plasmid prf-irdel reduced luciferase activity by ~ fold. the deletion of ppt sequence in plasmid prf-irdel alone, or the simultaneous deletion of ppt ppt and the intervening sequence in prf-irdel reduced the luciferase activity to background. this effect was further confirmed by western blotting using deletion constructs. in all constructs, the firefly luciferase gene was replaced by the egfp coding region. the wild type ires construct pre-ir and the deletion constructs, pre-irdel pre-irdel and pre-irdel along with the control vector pre were transiently transfected into the n2a cells. fourty eight hours post transfection cytosolic cell extracts were prepared and μg of the total protein was separated by 10% sds-page. immunodetection with the anti-gfp antibody showed an enhanced expression of egfp in the construct pre-ir with the wild type ires element as compared to the control vector pre. the deletion of the ppt motif induced a moderate decrease in the expression of egfp as compared to pre-ir. the deletion of the ppt motif alone or a deletion of both ppt and ppt reduced egfp expression below the detection limit. this finding confirmed the profound effect of ppt on ires activity consistent with the result obtained by the reporter assay described above. polypyrimidine tract binding protein plays an essential role in the ires activity mediated by ppt and ppt2 the polypyrimidine tract binding protein is known to bind to polypyrimidine tracts. we assumed a role of ptb in binding to the two polypyrimidine tracts and thereby modulating ires activity. this hypothesis was tested by overexpression of the human ptb in n2a cells together with the wild type ires or ppt deletion constructs. after transient transfection into n2a cells, rennila and firefly luciferase activity was measured and ratios calculated. luciferase readings showed that co-transfection of the vector pc1-ptb encoding the exogenously expressed huptb with the wild-type ires construct prf-ir stimulated luciferase activity ~ fold as compared to control vector prf. similarly, co-transfection of the ppt deletion construct, prf-irdel and pc1-ptb, showed a ~ fold increase in the luciferase activity. in contrast, simultaneous expression of the ppt deletion constructs prf-irdel and prf-irdel with huptb did not affect luciferase activity. these results demonstrate that ppt is crucial for ptb mediated ires activity, while ppt seems to play a subsidiary role. a direct correlation between the expression of ptb and the activity of the ires element was substantiated by western blots using the egfp containing di-cis constructs. the wild type ires construct pre-ir and the deletion constructs were transiently co-transfected with the pc1-ptb vector into n2a cells. cell extracts were prepared hrs after transfection and μg of the total protein was separated on 10% sds gel. immunodetection with anti-gfp and anti-ptb antibodies showed that the huptb over-expression along with the wild type ires element resulted in an enhanced expression of egfp as compared to endogenous expression of ptb. co-expression of huptb and the ppt deletion construct promoted an increased egfp expression, which appeared only slightly reduced when compared to the wild type ires element. the deletion of the ppt motif, however, led to a profound reduction of egfp protein expression. the double mutant lacking ppt and ppt was indistinguishable from the control vector pre demonstrating that both ppt motifs are necessary to mediate the full ptb effect, but with different efficiency. polypyrimidine tract i and polypyrimidine tract ii are important for the ires mediated expression of the p11-ct of cx the importance of ptb and polypyrimidine tracts for the expression of the ires-dependent translation of the carboxy-terminal fragment of cx was also analyzed using the mutant deficient for both polypyrimidine tract binding sites. the mutated construct and the wild type cx -egfp construct were transiently transfected into n2a cells either in presence or absence of over-expressed huptb. hrs post-transfection cytosolic cell extracts were prepared and μg of the total protein was separated on 10% sds gels. immunodetection with anti-gfp showed that the deletion of the ppt and ppt motif reduced the expression of p11-ct as compared to the wild type construct. furthermore, over-expression of huptb with the wild type cx construct resulted in the expected enhanced expression of p11-ct, while the over-expression of huptb with the pptdel mutant promoted p11-ct expression to levels below that of the wild type construct. a specific ribonucleic-protein complex assembles on the cx ires element we used the rna electromobility shift assay to determine whether cellular proteins recognize the cx ires element. for this purpose, a radiolabeled rna probe was incubated with a s cytosolic n2a protein extract. as shown in fig. 5a the cytosolic s extract retarded the migration of the rna probe, leading to the formation of a single dominant rna-protein complex when compared to the control reaction without protein. formation of this complex was effectively competed by the inclusion of a 50-fold molar excess of homologous unlabeled competitor rna. additionally, uv cross-linking experiments were performed to further characterize the rna-protein complexes that assemble at the ires element. when the wild type ires probe, or its deletion mutants were incubated with the s cytosolic n2a protein extract and uv crosslinked several distinct rna-protein complexes were detected after sds-page. the crosslinked complexes had apparent molecular masses in the range from to kda. a prominent band showed an apparent molecular weight of ~ kda. the formation of the cross-linked rna-protein complexes was prevented by the inclusion of a fold molar excess of unlabeled homologous competitor rna. when the deletion mutants pptdel pptdel and pptdel were applied a reduction of the protein complex at around ~ kda became apparent, indicating that the deletion of the polypyrimidine tracts weakens the binding of a major rna binding protein. purified gst-ptb fusion protein is able to bind the ires element the prominent rna-protein complex of ~ kda suggested that ptb with an apparent molecular weight of kda constitutes the protein binding to the ires element. we evaluated this assumption with a recombinant gst-ptb fusion protein, which was purified to homogeneity and subjected to rna-emsa. the wild-type ires rna probe and the deletion mutants were incubated with gst alone or with the gst-ptb fusion protein. as shown in, gst-ptb was able to retard the migration of the rna probe while gst alone did not show any effect. the formation of the gst-ptb rna complex was prevented by inclusion of fold molar excess of homologous unlabeled competitor rna. the rna-emsa data were further confirmed by uv cross-linking of the gst-ptb fusion protein to the wild type ires rna, which resulted in the formation of a single rna-protein complex of ~ kda. this complex was effectively competed by adding fold molar excess of unlabeled homologous rna. no rna-protein complex was formed by uv cross-linking gst alone to the ires element, which excludes that the gst protein itself has rna binding properties. the pptdel ires rna showed a reduced binding when compared to the wild type ires rna. this effect was even more pronounced when the pptdel and pptdel mutants were subjected to cross-linking. ptb interacts with zfcx mrna in vivo under physiological conditions to prove that ptb interacts with the cx mrna in living cells under physiological conditions, ribonucleoprotein immunoprecipitation assays were performed. n2a cells were transiently transfected with a cx expression vector. hrs post transfection cells were harvested in pbs and incubated with formaldehyde to form stable rna-protein complexes. cells were lysed and ptb was immunoprecipitated from cell lysates using ptb specific antibodies. the rna was extracted, reverse transcribed to generate cdna and subjected to polymerase chain reaction with primers specific to the cx coding region. the specificity of this approach was proven using β-actin antibody coated beads and a void control with beads only. as shown, a specific dna band of about bp corresponding to the full length cx coding region was pcr amplified using the cdna obtained from the immunoprecipitated rna. to further confirm the specificity of the immunoprecipated rna, nested primers specific for the carboxy-terminal domain of cx were employed that led to the amplification of the expected carboxy-terminal fragment of ~ bp. no amplicons were found in controls using the β-actin loaded and void beads. thus, the rnp-ip assay provides additional evidence that ptb is able to interact with the cx mrna in living cells under physiological conditions and strengthens our assumption that ptb constitutes a crucial factor involved in activating ires mediated translation of this neuronal connexin. discussion and  internal ribosome entry sites are rna complexes with extensive secondary structures. several conserved motifs have been described to be essential in the regulation of ires activity among which polypyrimidine tracts are well documented. polypyrimidine tracts are involved in the regulation of mrna translational mechanisms and the importance of oligopyrimidine sequences have already been reported upstream of aug initiation codons in picornaviruses and in the hepatitis c virus. in continuation of previous studies, we analyzed the internal ires element of cx for sequence motifs of trans-acting factor, which are important for the function of ires activities. sequence analyses showed the presence of two stretches of polypyrimidine tracts, ppt and ppt flanking the in frame ires sequence of cx. to investigate the influence of these polypyrimidine tracts on the activity of the ires element, we deleted ppt and ppt either alone or in combination. subsequent reporter assays and western blot analyses showed that deletion of ppt had an appreciable effect on the ires activity, while deletion of ppt results in complete loss of ires activity. this result indicates that the bp stretch of ppt serves an important function in determining ires activity of cx albeit by altering the rna fold. in addition to their requirements for eukaryotic initiation factors, the efficiency of most ires elements is augmented by non-canonical initiation factors know as itafs. since our initial results indicated an involvement of polypyrimidine tracts in defining the ires activity of cx, we investigated the role of the polypyrimidine tract binding protein. the ires activity of the wild type ires element and its deletion mutants was analyzed in the presence of endogenous levels of ptb or upon overexpression of the protein by transfection with huptb. overexpression resulted in a significant increase of ires activity of the wild type ires element. deletion of the ppt element led to a significant reduction as compared to the wildtype, but the ires element was still responsive in particular when exogenous huptb was added. deletion of ppt alone or in combination with ppt revealed a complete loss of responsiveness. our results suggest that the polypyrimidine tract is crucial for the ires activity and that ptb conveys a role in the regulation of the ires activity of cx. a plausible explanation for the critical role of ppt in the ires activity is that it binds to additional crucial trans-acting factor, which are important for the recruitment of the ribosomal translational machinery. alternatively, this tract is important for the specific rna secondary structure and the role of ptb seems to stabilize the active confirmation of the ires element through binding to the rna scaffold. interestingly, the secondary structure prediction using the rna folding algorithm mfold of the wild type ires element and its deletion mutants revealed that the wild type ires of cx has an extended stem-loop structure with semi-conserved y-like configuration, described also for ires elements found in some picornaviruses. deletion mutation of ppt showed overall a similar structure when compared to the wild type rna fold with a minor loss of the small stem loop. in contrast, the deletion mutant ppt showed a complete remodeling of the structure, which was predicted to be energetically less stable as compared to the wild type ires rna fold. the ptb protein is very well known in regulating ires activity of both viral ires and cellular ires elements. these data are of particular interest keeping in mind that ptb is primarily a nuclear protein, where it plays a role in regulation of splicing events of eukaryotic mrnas. to become functional in ires related activities, ptb needs to shuttle from the cell nucleus to the cytoplasm. recent evidence on the involvement of ires elements in internal translation of biologically active protein domains makes it essential that this processes need to be regulated. shuttling events of ptb from the cell nucleus to the cytoplasm can be one step where regulation by internal and external factors becomes effective. one such mechanism has been described recently. in this study, protein kinase a phosphorylation was found to modulate shuttling of ptb by phosphorylation of a particular serine residue. this modification results in an increase of the cytoplasm-directed transport of ptb from the nucleus and couples the pka pathway with translocation events of ptb. in the context with our recent findings, which demonstrated expression of an ires driven carboxy-terminal fragment of cx and its translocation to the nucleus of horizontal cells a link between ptb shuttling and regulated expression of this fragment is challenging.. due to the prominent role of gap junction coupling in horizontal cells in primary processing of visual stimuli and its dependence on dopamine regulation, it will be of considerable interest to examine whether a camp dependent mechanism involving dopamine affects ptb shuttling and contributes to the strength of intercellular horizontal cell coupling. alternatively, retinoic acid, which exerts a profound effect on synaptic plasticity of horizontal cells and involves pka phosphorylation to execute its signal transmission potency may comprise another candidate, which could operate through the indicated mechanism. aside of the identification of ptb as a protein binding to the cx ires element further binding proteins covering the range of ~ to kda were identified by uv crosslinking experiments. some of them are in the size range of proteins, which have been described to bind to other ires elements. it remains to be established whether these proteins constitute further component of this complex, and whether they are also involved in functional regulation of the cx ires element. in summary, we demonstrate that the activity of the internal ires element of the horizontal cell specific gap junction protein cx is modulated by two polypyrimidine tracts. we further provide evidence that the polypyrimidine binding protein works as a non-canonical factor in the regulation of the ires activity. the physiological meaning of the cx ires activity and in consequence the generation of the p11-ct protein in horizontal cell function awaits further clarification. 
selective deletion of ppt results in an appreciable decrease of the ires activity, while the deletion of ppt results in a complete loss. this observation is of considerable importance and may provide initial insight into molecular-functional relationships of electrical coupling in horizontal cells. two stretches of polypyrimidine tracts designated ppt and ppt which influence the ires activity of this neuronal gap junction protein were identified. rna-emsa and uv-cross linking experiments showed that protein complexes bind to this ires element, of which the polypyrimidine tract binding protein was identified as one of the interacting partners with influence on ires activity. our findings indicate that the activity of the ires element of the neuronal gap junction protein cx is subject of regulation through flanking polypyrimidine tracts, and that the non-canonical trans-activation factor ptb plays an essential role in this process. we characterized the ires element of cx in terms of sequence elements necessary for its activity and protein factor, which may play a role for its function. 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. these results indicate that ptb conveys a role in the regulation of the ires activity of cx.
 connexin 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.  we characterized the ires element of cx in terms of sequence elements necessary for its activity and protein factor, which may play a role for its function. two stretches of polypyrimidine tracts designated ppt and ppt which influence the ires activity of this neuronal gap junction protein were identified. selective deletion of ppt results in an appreciable decrease of the ires activity, while the deletion of ppt 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 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 cx.  our findings indicate that the activity of the ires element of the neuronal gap junction protein cx 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. 