- Research article
- Open Access
IRES-mediated translation of the carboxy-terminal domain of the horizontal cell specific connexin Cx55.5 in vivo and in vitro
© Ul-Hussain et al; licensee BioMed Central Ltd. 2008
- Received: 06 July 2007
- Accepted: 27 May 2008
- Published: 27 May 2008
Changes of the interneuronal coupling mediated by electrical synapse proteins in response to light adaptation and receptive field shaping are a paramount feature in the photoreceptor/horizontal cell/bipolar cell (PRC/HC/BPC) complex of the outer retina. The regulation of these processes is not fully understood at the molecular level but they may require information transfer to the nucleus by locally generated messengers. Electrical synapse proteins may comprise a feasible molecular determinant in such an information-laden signalling pathway.
Connexin55.5 (Cx55.5) is a connexin with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the PRC/HC/BPC complex in form of hemichannels where light-dependent plasticity occurs. Here we provide evidence for the generation of a carboxy-terminal domain of Cx55.5. The protein product is translated from the Cx55.5 mRNA by internal translation initiation from an in-frame ATG codon involving a putative internal ribosome entry site (IRES) element localized in the coding region of Cx55.5. This protein product resembling an 11 kDa domain of Cx55.5 is partially located in the nucleus in vivo and in vitro.
Our results demonstrate the generation of a second protein from the coding region of Cx55.5 by an IRES mediated process. The nuclear occurrence of a fraction of this protein provides first evidence that this electrical synapse protein may participate in a putative cytoplasmic to nuclear signal transfer. This suggests that Cx55.5 could be involved in gene regulation making structural plasticity at the PRC/HC/BPC complex feasible.
- Enhanced Green Fluorescent Protein
- NIH3T3 Cell
- Internal Ribosome Entry Site
- Horizontal Cell
- Electrical Synapse
Direct communication via gap junctions between cells is important for coordinated cellular activity. Connexins play a central role in this biological function and contribute to tissue homeostasis and electrical coupling by forming communicating channels between adjacent cells. In general, the significance of connexin expression has been attributed to gap junction coupling. However, recent evidence suggests that connexins may play other roles than being the integral part of gap junction channels. In fact, connexins and/or processed connexin fragments may influence important biological functions like regulation of cell growth [1–3] and resistance to cell death  by mechanisms that do not require gap junction communication [5–8] but necessitate cytoplasm to nucleus signalling by locally generated messengers.
In brain tissues interneuronal signalling is conveyed by chemical and electrical synapses, the latter being formed by gap junctions. Extensive data exists on the nature of locally generated messengers which target to the nucleus serving important function for activity-dependent control of neuronal gene expression during chemical signalling transmission [9–11]. Evidence for mechanisms that may play a similar role is entirely missing for electrical synapses.
We chose the photoreceptor/horizontal cell/bipolar cell (PRC/HC/BPC) complex of the retina to screen for such mechanism for the following reasons: (i) The PRC/HC/BPC complex is endowed with connexins either in form of hemichannels and/or of paired gap junctions . (ii) The PRC/HC/BPC complex exhibits a remarkable restructuring in response to ambient light exposure, and can be regarded as a model for long-term activity-dependent electrical synapse plasticity [13–15]. (iii) HCs are unique insofar as they reveal a highly restricted pattern of connexin expression. Mouse HCs express Cx57 the orthologue of the human Cx59 . In zebrafish the expression of two related connexins has been described: Cx52.6 and Cx55.5 [17, 18] with the latter accumulating in HC dendrites which are involved in the activity dependent plasticity of the PRC/HC/BPC complex .
All connexin isoforms are presumed to have similar topology, which has been deduced from limited proteolysis and the application of site directed antibodies . The NH2-terminal and the COOH-terminal domain are localized in the cytoplasm and are connected by four transmembrane domains, two extracellular loops and a cytoplasmic loop. Recent evidence indicates that the carboxy-terminus of one of the most abundant connexins (Cx43) may be involved in gene regulation by either interacting with growth regulators [20, 21] or nuclear-translocation of processed carboxy-terminal domains .
The coding region of the zebrafish Cx55.5 lies in a single main exon consisting of 1497 bp  located downstream of two promoters . It shares the same topology with other connexins and exhibits an exceptionally long COOH-terminal tail of ~288 amino acids characterized by an unusual clustering of serine residues. Thus Cx55.5 provides a likely candidate to look for activity-dependent processing of signalling domains. Evidence that some connexins are regulated at the posttranscriptional level initially derived from studies with Cx26, Cx32 or Cx43. The 5'-UTRs of these connexins contain an internal ribosome entry site (IRES), enabling translation in a cap-independent manner [24–26]. Additionally, it has been demonstrated that the Xenopus Cx41 5'-UTR contains three upstream open reading frames (uORFs) that significantly repress translation in young embryos .
In the present report, we provide evidence for a unique way of an internal expression of a carboxy-terminal domain of Cx55.5, mediated by a putative IRES element present within the coding region. Activation of this IRES element elicits the processing of an 11 kDa carboxy-terminal fragment (p11-CT) which could be detected in the cytoplasm and the nucleus of the retina in vivo and in heterologous expression systems in vitro suggesting a cytoplasm to nucleus signalling mechanism through a messenger generated from an electrical synapse protein.
Cx55.5 immunoreactivity in the nucleus of horizontal cells and identification of a candidate protein
Since the antibody applied was generated by immunization with a fusion protein comprising the entire Cx55.5 CT domain (nt629–nt1497) it was reasonable to speculate that the ~16 kDa protein derived from the CT of Cx55.5, and may comprise the source for the nuclear reactivity. This assumption was experimentally confirmed using fractionated protein extracts deriving from the retina of adult fish (Fig. 1H). As expected the full length Cx55.5 protein product resembling an integral membrane protein was restricted to the cytoplasmic cell fraction composed of soluble and membrane proteins (upper lane). The ~16 kDa protein product was found in the cytoplasmic and nuclear extract (middle lane). Note that β-actin is depleted in the nuclear fraction as expected (bottom lane). Thus, the ~16 kDa fragment fulfilled the criteria as candidate protein for the Cx55.5 immunoreactivity observed by the immunohistochemical localization.
Full length Cx55.5 and a portion of its carboxy-terminal domain are co-translated
The carboxy-terminal protein p11-CT is translated from the Cx55.5 transcript by internal translation
An IRES element in the coding region of Cx55.5 is responsible for the expression of the p11-CT protein
To further delineate the putative IRES element, the pRF-IR1 construct was truncated by removing a ~200 bp fragment from the 3'end of the pRF-IR1 construct. This new construct pRF-IR2 (Fig. 4A) with a shortened Cx55.5-CT domain (nt631 to nt990) along with the construct carrying the entire fragment (pRF-IR1) and the control pRF-Di-cis vector were transiently transfected into N2A, HeLa and NIH3T3 cells. Subsequent luciferase activity determination indicated a substantial overall increase of IRES activity. The increase over control levels was ~35 fold in HeLa, ~34 fold in N2A, and ~77 fold in NIH3T3 cells (Fig. 4B). This observation indicates that the DNA sequence immediately upstream of the in frame ATG codon (nt1201) exhibits a regulative function on the IRES activity.
Increased expression of the second cistron in the Di-cistronic assay is due to IRES activity and not a cryptic promoter
The p11-CT protein is not expressed from a monocistronic mRNA
Next we performed western blot analysis of N2A cells expressing the control vector pRE-Dis cis or pRE-IR2 (see Additional file 2). We assumed that a cryptic promoter activity should lead to a reduced RLuc protein and increased EGFP protein expression when EGFP is translated from a monocistronic message. In contrast, when both proteins are generated from a single mRNA we expected that RLuc expression should be unaffected but EGFP expression substantially increased in the presence of the IR2 element. As shown, simultaneous detection of the Renilla luciferase and EGFP showed that the translation of the second cistron was greatly enhanced when the IR2 element was included in the bicistronic vector. In line with the latter assumption the expression of the first cistron appeared unchanged.
In summary, the results obtained by independent methods conclusively suggest that p11-CT is not expressed from a monocistronic mRNA.
The p11-CT product is partially located in the nucleus in vitro
In summary, we provide evidence that an IRES-mediated molecular process may account for the generation of a small CT domain of the HC connexin Cx55.5, and that this protein product is potentially capable to translocate into the nucleus both in vivo and in vitro.
In this report, we describe that the internal translation involving a putative IRES element located in the coding region of connexin Cx55.5 leads to the formation of two alternative protein products in vivo and in vitro. Our initial observation of low levels of p11-CT immunoreactivity inside the nuclei of horizontal cells in vivo has led us analyze putative molecular mechanisms underlying the generation of this Cx55 protein fragment.
By performing sequence analyses of the coding region of Cx55.5 we found several in frame ATG codons. Three candidate in-frame ATG codons in the long carboxy-terminal tail of Cx55.5 were taken into consideration. The mutation analysis confirmed the ATG at nucleotide position 1201 as translation initiation site giving rise to a protein product with a theoretical molecular weight of 11 kDa in case of internal translation.
On the basis of the above observations we hypothesized that the generation of the p11-CT is achieved by an IRES element in the coding region of Cx55.5. Since IRES mediated activities have been scrutinized recently [31, 32] we performed stringent control experiments to rule out activities of cryptic promoters, RNAse cleavage or cryptic splicing. To date the number of IRES elements found in connexin genes is limited and those IRES elements identified are restricted to the 5'UTR of Cx43, Cx32 and Cx26 [24–26]. More elements have been reported in the 5'UTR of other eukaryotic genes [33–38]. The existence of IRES elements in the coding region of eukaryotic genes is still a rare observation with only a few reports in the literature [39, 40], where in some examples similarly to the p11-CT the carboxy-terminal domain has been described to be internally translated .
Supporting data on a nuclear presence of p11-CT come from our in vitro studies which included confocal laser imaging and western blots of nuclear extracts. Both demonstrated nuclear localization of the p11-CT fusion protein when transfected in NIH3T3 cells. Different from the transfected cells the abundance is less under in situ conditions and restricted to a subpopulation of horizontal cells. This, however, is as expected, since the amount of physiologically expressed and hence translocated protein must be regarded to be lower in the intact retina as compared to the over-expressing cell lines.
A nuclear localization of a carboxy-terminal domain of a connexin has been already reported in case of Cx43 . A nuclear translocation of carboxy-terminal domains would be of paramount importance as far as gap junction biology is concerned. The existence of a molecular mechanism initiating an internal expression of CT fragments could provide first insight into connexin properties not readily explainable by its channel forming properties. A separate expression of biologically active CT-domains and their nuclear translocation can endow gap junction proteins with the capability of modulating gene expression directly in response to changes in physiological activities and/or pathophysiological challenges.
The biological significance of a separate expression of a CT-domain of Cx55.5 via IRES mediated internal translation is still to be established. An argument in favour of a possible functional link between expression of Cx55.5 and retinal activity derives from the observation that both Cx55.5 isoforms are strictly expressed in HCs. Most importantly, the Cx55.5 full length protein is known to accumulate exactly at those sites known to undergo a dramatic light dependent reorganization of the postsynaptic compartment of the HC/RC/BPC complex in form of reshaping of the HC dendritic spinules after light withdrawal [12, 42–44]. Since these sites have been proven to be responsible for changes of functional responsiveness of the HC/RC/BPC complex accompanying dark adaptation , a signalling pathway involving Cx55.5 is suggestive. At present the molecular mechanism involved is unclear but our recent identification of the RNA binding polypyrimidine tract binding protein (PTB) as interaction partner at the putative Cx55.5 IRES element (M.U-H, unpublished data) is suggestive for a mechanism involving the cAMP/PKA signalling pathway . Furthermore, the basic properties of the p11-CT domain with a calculated pI of >10 may resemble properties comparable to the basic DNA binding domains of some transcription factor gene families . Therefore, future studies need to uncover whether the subcellular localization and/or potential DNA binding capabilities of p11-CT underlay mechanisms similar to C/EBPdelta  or Gli1 , two nuclear transcription factors known to shuttle between cytoplasm and nucleus upon activation of a PKA-driven pathway.
The described activity of an IRES sequence and the translation of the p11-CT fragment of Cx55.5 may resemble a novel regulative mechanism which governs the plasticity of the PRC/HC/BC complex. It awaits further exploration to unearth the mechanistic background which links the functional with the molecular side.
Zebrafish were kept at 28°C in aerated tanks filled with tap water circulating through an bacterial filter systems. The fish were kept on a 14 hour ON/10 hour OFF light-dark cycle. All animal experiments were carried out according to the guidelines of the German Animal Protection Law in its present version (1998) and under the responsibility of the ethical committee of the Royal Netherlands Academy of Arts and Sciences acting in accordance with the European Communities Council Directive of 24th November 1986 (86/609/EEC). All experiments including animals were kept to the necessary minimum.
Full length Cx55.5 (nt1–nt1497) was obtained by PCR amplification of the coding region from a genomic clone (GI:77747480) using primer pair S1 and AS1 (Additional File 5). The PCR product was ligated in-frame into pEGFP-N3 (BD Biosciences Clontech, CA, USA) and sequence confirmed. All other plasmid constructions summarized in the Additional file 4 derived from this construct and were cloned using standard recombinant DNA techniques. The Di-cistronic vector pRF-Di cis comprising the Renilla luciferase as first cistron, the Firefly luciferase as second cistron, the human globin gene intron and stable hairpin structures has been described . The plasmid was a kind gift from Dr. Rudolf Werner (Department of Biochemistry and Molecular Biology, University of Miami, School of Medicine). The pGL3-control vector was obtained from Promega (Promega Corporation, Madison, WI, USA).
Cell culture, transfection and reporter-assays
HeLa, NIH3T3 and N2A cells were purchased and maintained in cell culture as recommended by the ATCC (LGC Promochem GmbH, Wesel Germany). For determination of IRES activity, 2 × 104 N2A cells were plated in 96 well flat bottom plates (BD Biosciences), transiently transfected using 100 ng plasmid DNA and the Effectene® transfection protocol (Qiagen, Hilden, Germany). 48 hours after transfection, luciferase activity was measured in an Orion II Micro plate Luminometer (Berthold Detection Systems, Pforzhein, Germany), using the Dual-Luciferase Reporter assay system (Promega Corp., Madison, Wisconsin, USA). IRES activity was expressed as the ratio of Firefly luciferase/Renilla luciferase (FLuc/RLuc) with the activity of the control vector (pRF Di-cis.) set to 1. Each experiment was performed 5 times with all constructs in triplicates. Data are expressed as mean ± SEM.
Generation of a Cx55.5 specific polyclonal antibody
A polyclonal Cx55.5 antibody was generated by immunization of Chinchilla rabbits. The antibody was generated against the carboxy-terminal domain of Cx55.5 (amino acids 231–498) which was expressed as a GST fusion protein in the E. coli strain BL21. The serum was affinity purified using GST-Cx55.5 crosslinked to a HITrap-Sepharose column matrix and eluted as recommended by the manufacturer (GE Healthcare UK Ltd, Little Chalfont Buckinghamshire, UK). The specificity of the antibody was confirmed by western blotting and immunocytochemistry using preimmune serum and/or preabsorption with GST-Cx55.5 as controls. Cross reactivity with the second horizontal cell connexin (Cx52.6) was excluded by absorption to GST-Cx52.6 as described .
Western blot analyses
For western blot analyses of transfected cell lines, 2 × 105 N2A or NIH3T3 cells were grown in 12 well plates (BD Biosciences). Transient transfections were performed using 300 ng plasmid DNA and the Effectene® transfection protocol (Qiagen). 48 hours after transfection, cytosolic and nuclear extracts were prepared according to the manufactures protocol (Active Motif Nuclear Extraction Kit, Rixensart, Belgium). 20 μg of each protein fraction was separated on 10% SDS PAGE and processed as described previously . In some experiments the detection antibody was replaced and the IRDye 680 Goat Anti-Mouse IgG detection antibody (1:20,000) used in combination with the Odyssey infrared detection system (LI-COR Biosciences, St. Lincoln, NE, USA).
For western blot analysis of adult zebrafish tissues total protein extracts were isolated from freshly dissected retina and brain by direct homogenization in denaturing Leammli buffer. Alternatively, cytosolic and nuclear protein fractions were generated with the identical kit described above. 20 μg protein was separated by 15% SDS-PAGE, transferred to 0.2 μm NC membrane (Protran BA83, Schleicher & Schüll BioScience, Dassel, Germany) and processed as described above. Primary antibodies were diluted 1:2,000 (anti-GFP; Roche Applied Science, Mannheim, Germany), 1:1,000 (anti-FLAG; Sigma-Aldrich, St. Louis, USA) and 1:7,500 (anti-β actin; Sigma-Aldrich). The primary anti-Cx55.5 antibody was used at 1 μg/μl.
RNA expression analysis
For expression analysis, 2 × 106 N2A cells were seeded in 6-well plates (BD Biosciences). RT-PCR was performed as a test for mRNA splicing in N2A cells following DNA transfection of the control Di-cis vector and the IRES containing Di-cis constructs. Total RNA was isolated as described above from transiently transfected N2A cells. First strand cDNA synthesis was carried out as described  and PCR amplified using the primer pairs DI1/DI3 and DI2//DI3 (see Additional file 5).
Immunohistochemistry and immunoelectronmicroscopy
Eyes were isolated from cervically transected fish and processed for immunohistochemistry and immuno electron microscopy as described . Confocal image analysis was performed using the LSM 510 Meta system (Zeiss), equipped with argon and HeNe lasers, 40× (NA 1.4) and 63× (NA 1.4) oil objectives and the LSM 510 Meta software as described .
The work was sponsored by grants from the Deutsche Forschungsgemeinschaft to R. D. and G. Z. (DFG 292/11-3 and SFB 509), and a travelling grant from the International Graduate School of Neuroscience, Bochum, Germany to M.U.H. We would gratefully mention the invaluable support of Marian Kremer who dedicated most of her active working life to the cloning and characterization of zebrafish connexin genes. Furthermore, we thank Dr. D. Krause-Finkeldey and Dr. K. Ladage for help with the antibody production.
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