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J. Biol. Chem., Vol. 279, Issue 16, 15877-15887, April 16, 2004
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From the
School of Biomedical Science, University of Queensland, Brisbane, Queensland 4072, Australia, the Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom, and the ¶Victor Chang Cardiac Research Institute, Sydney, New South Wales 2010, Australia
Received for publication, December 12, 2003 , and in revised form, January 29, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The expression of CFTR transcripts and cAMP-activated chloride currents in the heart have been demonstrated for humans and simians (18), rabbits (19), guinea pigs (20, 21), and cats (22), but are undetectable in murine (23) and canine hearts (24). Previous studies have shown that CFTR transcripts expressed in the heart are alternatively spliced, resulting in the loss of exon 5 (25). We have previously demonstrated that CFTR mRNA is expressed in an epicardial (higher) to endocardial (lower) gradient across the left ventricular free wall (LVFW) of the rabbit heart, coinciding with a 2.5:1 gradient in the cAMP-activated chloride current density in ventricular myocytes (26). Furthermore, this epicardial to endocardial gradient in CFTR expression is developmentally regulated, appearing in the first postnatal week (27), and lost in hypertrophic and failing hearts (28). The cardiac distributions of CFTR mRNA and functional channels are consistent with a role in the maintenance of the normal epicardial to endocardial gradients of ventricular repolarization and action potential duration in the heart (14, 26). In addition, the overall reduction and loss of the gradient of CFTR expression during hypertrophy could contribute to delayed ventricular repolarization (29) and loss of the gradient of repolarization in hypertrophied hearts (30), both of which are known to be arrhythmogenic (31). Indeed, it has been shown that some CF patients exhibit an increased risk of ventricular arrhythmia (32, 33); however, it is difficult to distinguish between a primary genetic cause due to the loss of CFTR expression and secondary effects as a result of pathological manifestations in the pulmonary system.
In this study, we investigated the regulation of temporal, regional, and pathological changes in CFTR expression in the rabbit heart. We show that the majority of CFTR transcripts expressed in the heart initiate at unique transcription start sites and include novel alternative 5'-exons that replace the traditional CFTR exon 1. These alternative 5'-exons encode (a) a series of short upstream open reading frames (uORFs) in the 5'-untranslated region (5'-UTR), resulting in post-transcriptional regulation of CFTR expression, and (b) a unique translation initiation codon, in-frame with the main CFTR open reading frame (ORF), which results in a CFTR polypeptide with a distinct amino terminus. Modulation in the levels of these cardiac-specific CFTR transcripts is responsible for the temporal, spatial, and pathological changes in CFTR expression observed in the heart. Finally, the distal localization of these cardiac-specific, alternative 5'-exons upstream of the traditional CFTR exon 1 suggests the presence of a distinct promoter region directing CFTR expression in the heart.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5'-Rapid Amplification of cDNA Ends (5'-RACE)—CFTR transcription start sites were identified by 5'-RACE as previously described (11). Total RNA (2 µg) was reverse transcribed using a CFTR exon 6 reverse primer (Table I). First-strand cDNAs, tailed with dCTP, were subject to two rounds of hemi-nested PCR using reverse primers to exons 4, 3, or 2 and an anchor primer to the dC tail. Amplicons were sequenced directly and subcloned into pLITMUS 28 (New England Biolabs).
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-32P]dCTP (3000 Ci mM, Amersham Biosciences; DECAprime II system, Ambion). Total RNA was electrophoresed on a 1% (w/v) denaturing agarose gel, transferred to a positively charged nylon membrane (GeneScreen Plus, PerkinElmer Life Sciences), incubated with labeled probe, and then washed as previously described (13). Autoradiography was with Hyperfilm MP x-ray film (Amersham Biosciences) for 5 days. TaqMan Quantitative PCR (qPCR)—Relative CFTR expression was quantified using cDNA primed with random hexamer (500 ng) with transcript-specific forward primers (exons –1C, –1B, –1A, 1, 4, and 5), reverse primers and probes (exons 2 and 6), all designed to cross at least one exon-exon boundary (Table I). The 5'-reporter and 3'-quencher dyes used for CFTR probes were 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively, whereas the 18 S rRNA probe used the VIC reporter dye (Applied Biosystems). Each qPCR reaction consisted of 1x TaqMan Universal PCR Master Mix (Applied Biosystems), forward and reverse primers (250 nM), probe (50 nM), and cDNA template (100 ng). Triplicate experimental CFTR and endogenous 18 S rRNA reactions were performed using three independent cDNA samples from different animals. Reporter dye fluorescence was detected using an Applied Biosystems Prism 7700 Sequence Detector, and data were analyzed using Sequence Detector software version 1.6.3 (Applied Biosystems). Quantification of CFTR gene expression, relative to 18 S rRNA, was performed using the comparative threshold cycle method according to instructions from the manufacturer (Applied Biosystems).
Construction of CFTR-Enhanced Green Fluorescent Protein (eGFP) Fusion Constructs and Cell Culture—CFTR cDNA fragments containing each alternative or traditional exon 1 sequence plus exon 2 were ligated to the eGFP cDNA (Clontech) and the entire fragment subcloned into the pIRESneo mammalian expression vector (Clontech). Chinese hamster ovary (CHO) cells (ATCC, CRL-9096; courtesy of Dr. Yu Lu, University of Cambridge, Cambridge, United Kingdom) were grown in Iscove's modified Dulbecco's medium, 10% (v/v) fetal bovine serum, 4 mM L-glutamine, 18 mM sodium bicarbonate, 0.1 mM hypoxanthine, and 16 µM thymidine (Invitrogen). Triplicate wells of 60% confluent CHO cells were transiently co-transfected with 1 µg each of CFTR-eGFP and control enhanced blue fluorescent protein (eBFP) constructs, using LipofectAMINE according to the instructions from the manufacturer (Invitrogen). After 72 h of incubation, cells were trypsinized and transferred to a Thermo-Fast 96 black PCR plate (Abgene). Enhanced GFP and eBFP fluorescence was measured using a SPECTRAmax GEMINI-XS spectrofluorometer (36, 37). Prior calibration experiments determined the following optimal parameters: for eGFP, 
ex = 472 nm, em = 512 nm, and cutoff = 495 nm; and for eBFP, ex = 378 nm, em = 445 nm, and cutoff = 420 nm. To correct for differences in transfection efficiency, eGFP fluorescence values were normalized to eBFP signal. Background fluorescence (untransfected cells) was subtracted.
Bioinformatic and Statistical Analyses—Messenger RNA secondary structures were predicted using MFold version 3.1 (38, 39). CFTR amino-terminal hydropathy plots were generated using the Kyte and Doolittle algorithm (40). Protein secondary structures were predicted by PSIPRED version 2.4 (41). The charge distribution for the putative CFTR amino-terminal helix was predicted by helical wheel analyses using MacPlasmap Pro version 3.01. Statistical comparisons were made using Student's t tests (unpaired, two-tailed), and a p value of less than 0.05 was taken to indicate that a change in transcript level was of statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Transcripts that included exon –1A contain an AUG codon in-frame with the main CFTR ORF. Translation initiation from this AUG codon would result in CFTR protein with a novel amino-terminal region. Exons –1B and –1C did not contain an AUG codon in-frame with the main CFTR ORF. However, many upstream AUGs (uAUGs) were identified in the 5'-UTR of CFTR transcripts including exons –1C, –1B, or –1A, and a single uAUG codon was identified in the 5'-UTR of CFTRTRAD-139. These uAUGs defined the start of several uORFs, which were followed by translation termination codons and thus encoded short peptides. The presence of short uORFs in the 5'-UTRs of eukaryotic mRNA transcripts is indicative of post-transcriptional and translational regulation of gene expression (43). Data base comparisons of each putative short polypeptide sequence revealed no homology to known protein sequences.
Genomic DNA Structure and Transcriptional Control of CFTR Expression in the Heart—Rabbit genomic DNA (gDNA) spanning CFTR exon –1C to exon –1A, was amplified by long range PCR and the sequence compared with the human and mouse gDNA sequences (GenBankTM accession nos. AC000111 [GenBank] and AF162137 [GenBank] ; see Fig. 1C and supplemental data (available in the on-line version of this article) showing dot matrix comparisons of rabbit, mouse, and human sequences). We also compared CFTR traditional exon 1 and flanking sequences across the three species. In both exonic and intronic gDNA regions, human and rabbit sequences showed a high level of identity for both the CFTR exon –1C to –1A region and traditional exon 1 (Fig. 1C). However, only CFTR traditional exon 1 sequences showed any similarity between the mouse and the other species. The human homologues of rabbit CFTR exons –1C to –1A spanned a region located 6–9 kb upstream of the traditional human CFTR exon 1. Further, the short uORFs and the rabbit CFTR exon –1A AUG codon, that is in-frame with the main CFTR ORF, are conserved in the human genome. The presence of homologues of the rabbit alternative exon 1 sequences in the human genome, but not the mouse, is consistent with expression of CFTR in the hearts of many species, including the rabbit (Fig. 3A) (15) and human (18), but not in the mouse (tested by RNA in situ hybridization and two rounds of reverse transcription-polymerase chain reaction; data not shown). Only 2 kb of genomic DNA sequence upstream of rabbit CFTR traditional exon 1 is available, and the alternative CFTR exons –1A to –1C lie beyond that. Long range PCR between CFTR traditional exon 1 and exons –1A to –1C, using rabbit gDNA template, was unsuccessful. Therefore, the promoter region controlling the expression of CFTR exons –1C, –1B, and –1A is likely to be distally located and cardiac-specific.
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The predominantly expressed CFTR transcript in early rabbit cardiac development was CFTR–1B, with lower levels of the CFTR–1C and CFTR–1C/–1A transcripts (Fig. 2B). As development proceeds the expression of CFTR–1B transcripts decreased (p < 0.05), whereas expression of both CFTR–1C and CFTR–1C/–1A transcripts increased and became the predominant CFTR transcripts in the adult rabbit heart (p < 0.05 and p < 0.05, respectively). In contrast, the expression of CFTRTRAD-139 transcripts was low and static. In adult cardiac tissue, greater than 90% of total CFTR mRNA transcripts include the novel alternative exons (exon –1C, exon –1B, and exon –1C/–1A) described here. This contrasts with duodenum, where more than 95% of CFTR transcripts initiate from traditional CFTR exon 1.
Analysis of CFTR exon 5 alternative splicing showed equal levels of exon 5+ and exon 5- CFTR transcripts at embryonic and neonatal stages (Fig. 3C). In hearts from juvenile and adult animals, there were differential increases in expression of both CFTREXON5+, and CFTREXON5– isoforms. This resulted in CFTREXON5– transcripts being 3-fold more abundant than CFTREXON5+ transcripts in the LVFW of adult hearts (p < 0.05). This also contrasts with CFTR expression in adult duodenum, where over 90% of CFTR transcripts include exon 5.
The Expression of CFTR Transcripts Initiating at Exon –1C Is Primarily Responsible for the Epicardial to Endocardial Gradient across the LVFW—Epicardial (higher) to endocardial (lower) gradients across the rabbit LVFW have been shown for both cAMP-activated chloride currents and CFTR expression (26). Quantitative analysis of the differential distribution of each CFTR transcript in the adult LVFW shows that CFTR–1C and CFTR–1C/–1A transcripts, as well as alternatively spliced CFTREXON5– transcripts, are responsible for the epicardial to endocardial gradient of CFTR expression and function in the left ventricle (Fig. 3, A (i and iii) and B, p < 0.05 in all cases). In contrast, there were no substantial differences in epicardial versus endocardial expression of CFTR–1B, CFTRTRAD-139, or CFTREXON5+ transcripts (Fig. 3, A (ii and iv) and B).
This work has also identified a second, perpendicular gradient in CFTR expression along the apical to basal axis of the left ventricle (Fig. 3, A and B). All three cardiac-specific CFTR transcripts (CFTR–1C, CFTR–1B, and CFTR–1C/–1A) and both exon 5 alternatively spliced forms contribute to the apical to basal gradient (p < 0.05 in all cases). Collectively, these data show that CFTR expression is distributed in a radial pattern across the LVFW: highest at the apical epicardial surface and decreasing radially to the lowest point at the basal endocardial surface.
Cardiac Hypertrophy Causes the Loss of the Epicardial to Endocardial Gradient, but Does Not Alter the Apical to Basal Gradient of CFTR Expression—Cardiac ventricular hypertrophy is associated with a loss of repolarizing ion currents, including CFTR (28), and prolongation of the ventricular action potential duration (44). Here we show that the loss of the epicardial to endocardial CFTR expression gradient, in hypertrophic hearts, is caused by the preferential down-regulation of CFTR–1C and CFTR–1C/–1A transcripts (Fig. 3A; p < 0.05 in all cases), whereas CFTR–1B and CFTRTRAD-139 transcripts were unaffected (Fig. 3A (ii and iv)). Although cardiac hypertrophy resulted in the loss of the epicardial to endocardial CFTR expression gradient, the apical to basal gradient was unaffected.
Cardiac hypertrophy also differentially affected the expression of exon 5 alternatively spliced CFTR transcripts (Fig. 3B). The establishment of cardiac hypertrophy leads to a loss of the epicardial to endocardial gradient of CFTREXON5– transcripts, whereas CFTREXON5+ transcripts remain evenly distributed across the epicardial to endocardial axis. Further, both exon 5 alternatively spliced transcripts contribute to the apical to basal gradient in CFTR expression. The overall increase in CFTR expression during development reflects a preferential accumulation of CFTR–1C and CFTR–1C/–1A transcripts and a shift in exon 5 alternative splicing, such that CFTREXON5– transcripts predominate. This process is reversed in cardiac hypertrophy with preferential loss of CFTR–1C, CFTR–1C/–1A, and CFTREXON5– transcripts.
The Translation Initiation Codon in Exon –1A Supports CFTR Protein Production—In the absence of traditional CFTR exon 1, Carroll and co-workers (45) have provided evidence for translation initiation from downstream AUG codons, for example those present in exons 3 and 4. Of the CFTR transcripts expressed in the heart, only CFTRTRAD-139 and CFTR–1C/–1A transcripts contain an AUG codon upstream of exon 2 and in-frame with the main CFTR ORF. Both translation initiation codons, in exon –1A and traditional exon 1, equally match an optimal Kozak consensus sequence. Fig. 4A shows a comparison of the sequences surrounding each AUG codon and the Kozak consensus sequence (46). AUG codons present in exons 3 and 4 all showed significantly lower identity to the Kozak consensus sequence, suggesting translation initiation at AUG codons in exons 3 and 4 would be less efficient than at AUG codons in either exon –1A or traditional exon 1. Exon 3 and 4 AUG codons are the first AUG codons in-frame with the main CFTR ORF present in CFTR–1C and CFTR–1B transcripts.
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This experiment showed that efficient translation initiation does occur from the AUG codon identified in CFTR exon –1A, with an identical level of expressed protein produced by translation from the AUG codon in traditional CFTR exon 1 (Fig. 4B). There was no significant protein production from exon –1C and exon –1B fusion constructs. Protein expression from the AUG codons in either exon –1A or traditional exon 1 were 8-fold lower than eGFP alone (p < 0.01), suggesting that there may be elements within the 5'-UTRs of either CFTR–1C/–1A or CFTRTRAD-139 transcripts that may modulate the efficiency of translation initiation.
Upstream ORFs and 5'-UTR Secondary Structure Post-transcriptionally Modulate CFTR Expression—The presence of uAUG codons, distinct from the main ORF initiating methionine, in the 5'-UTR of most eukaryotic genes is unusual (47, 48). Inspection of the 5'-UTRs of the CFTR–1C, CFTR–1B, CFTR–1C/–1A, and CFTRTRAD-139 transcripts identified 16 putative translation initiation codons associated with uORFs. In the CFTR–1C/–1A transcript, we identified five uORFs in the 5'-UTR, whereas CFTRTRAD-139 encoded one uORF in the 5'-UTR. McCarthy and co-workers (43) have demonstrated that uORFs and mRNA secondary structure, such as stem-loops, can act alone or in combination to regulate translation initiation efficiency at downstream AUG codons.
Although both the CFTRTRAD-139/eGFP and the CFTR–1C/–1A/eGFP constructs supported translation, the efficiency of translation was reduced compared with eGFP alone. With uORFs and adjacent stem-loop secondary structures in both these CFTR 5'-UTRs, we shortened the 5'-UTRs to remove or reduce the number of uORFs and measured translation from the AUG of the main CFTR ORF. The CFTR–1C/–1A/eGFP construct was truncated to CFTR–1A/eGFP, reducing the number of uORFs from 5 to 3. Also, the 5'-UTR of the CFTRTRAD-139/eGFP construct was truncated by 65 nucleotides, removing the uORF and producing a 5'-UTR typical of CFTR transcripts expressed in duodenum (CFTRTRAD-74/eGFP).
Reduction in the number of uORFs located upstream of the in-frame AUG present in exon –1A caused a statistically significant (p < 0.05, Student's t test) 1.5-fold increase in protein production (Fig. 5A). However, the continuing presence of three uORFs is a likely reason for the still relatively low level of protein production. The removal of the only uORF in the 5'-UTR of traditional exon 1 resulted in a 3-fold increase in translation initiation at the AUG of the main CFTR ORF (p < 0.01), but this was still lower than that observed for control eGFP transfections.
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G values) of the stem-loop secondary structures were in the range of –20 to –30 kcal mol–1 (Fig. 5C). Messenger RNA stem-loop structures localized to the 5'-UTR, with similar calculated stabilities, have been shown to independently reduce translation initiation at a downstream AUG codon (49–51), but have a stronger inhibitory effect when localized immediately downstream of an uORF (43). Also, small differences in the sequence immediately surrounding the AUG codon, compared with the Kozak consensus, may contribute to the efficiency of translation.
Translation of CFTR–1C/–1A mRNA Results in CFTR Protein with a Unique Amino Terminus—As CFTR exon –1A splices directly to exon 2, the 17 amino acids encoded by traditional exon 1 are omitted during translation of CFTR–1C/–1A transcripts and 2 other amino acids, encoded by exon –1A, constitute the new CFTR amino terminus (Fig. 6A). The efficiency of translation of both the CFTRTRAD-139 and CFTR–1C/–1A transcripts is indistinguishable (Fig. 4B). As CFTR–1C/–1A transcripts are 
6-fold more abundant in adult heart, than CFTRTRAD-139 transcripts, it is likely that the majority of CFTR protein expressed in the heart is the curtailed amino terminus isoform reported here (CFTR–1C/–1A protein). We have used computational protein secondary structure and hydropathy analysis to predict possible differential functional roles of CFTR–1C/–1A protein.
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CFTR amino-terminal secondary structure was predicted by PSIPRED. Coinciding with the amino-terminal hydrophobic region described above (amino acids 11–26) is a putative helical structure (predicted with a high level of confidence) that spans the boundary between traditional exon 1 and exon 2 (Fig. 6C). Helical wheel analysis of this putative, hydrophobic helix predicts clustering of three positively charged residues (Fig. 6D). The removal of the amino-terminal half of this putative helix, as would be the case in the CFTR–1C/–1A isoform, is accompanied by a marked reduction in the confidence of prediction of the remainder of the helix. Both secondary structure and helical wheel predictions were used by Naren and co-workers (54) in the identification of a helix that has been confirmed to interact with syntaxin 1A and regulate CFTR channel activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Comparative phylogenetic analysis identified human homologues of both the rabbit exon –1C to –1A and traditional exon 1 CFTR regions, localized 10 kb upstream of human CFTR and at traditional exon 1, respectively. However, comparison with murine gDNA only revealed homology to traditional CFTR exon 1. These findings are consistent with species-specific, molecular, and functional distributions of CFTR expression in the heart, identified in humans (18) and rabbits (15, 25) but not in mice (23). Collectively, these findings suggest that CFTR transcription from exons –1C to –1A is cardiac-specific and controlled by a distinct, and previously unidentified, promoter. Additionally, this work provides support for the development of a rabbit model of cystic fibrosis, to allow investigation of some pathophysiological features of human CF disease that are not present in murine models, such as cardiac involvement in CF (56).
Spatial, developmental, and pathophysiological signals regulate CFTR expression in the heart, with the development of a left ventricular epicardial to endocardial gradient during the late fetal and neonatal periods, and loss of this gradient following hypertrophic stimuli (26–28). We have also identified an apical (higher) to basal (lower) gradient in CFTR expression that is unaffected by hypertrophy. This defines a radial pattern of CFTR expression across the left ventricle, correlating well with the spread of repolarization throughout the left ventricle. These findings further support the view that the cAMP-stimulated chloride current, encoded by CFTR, contributes to ventricular repolarization and differential action potential duration throughout the heart. The expression of multiple CFTR mRNA transcripts in the heart raises questions of differential regulation of specific transcripts by various signals.
During cardiac development there is a preferential accumulation of CFTR–1C, CFTR–1C/–1A, and CFTREXON5– transcripts. Concomitantly, there is a preferential loss of CFTR–1B transcripts. Additionally, the epicardial to endocardial gradient is primarily the result of CFTR–1C, CFTR–1C/–1A, and CFTREXON5– transcripts, whereas all cardiac-specific CFTR transcripts contribute to the apical to basal ventricular CFTR gradient. In contrast, CFTR transcripts initiated at traditional exon 1 are present at very low, static levels (less than 10%). Analysis of left ventricular CFTR gradients in the hypertrophic heart demonstrated a loss of the epicardial to endocardial gradient but not the apical to basal gradient, culminating in an overall decrease in the radial gradient of ventricular CFTR expression. Again, individual CFTR transcripts were differentially regulated, with a preferential loss of CFTR–1C, CFTR–1C/–1A, and CFTREXON5– transcripts, but no change in CFTR–1B and CFTRTRAD-139 expression. The overall increase in CFTR expression during heart development, and loss in cardiac hypertrophy is consistent with the "re-expression of fetal gene program hypothesis" to explain global gene expression changes in cardiac hypertrophy (57, 58). However, our findings extend that hypothesis to include differential effects on individual CFTR transcripts. This demonstration of the differential regulation of individual CFTR transcripts suggests further complexity in the regulation of CFTR expression, with mechanisms that allow transcript-specific interpretation of spatial, temporal, and pathologic signals.
We have identified CFTR regulatory mechanisms that involve both alternative splicing, and differential transcription start site and exon 1 usage. An important consequence of differential exon 1 usage is the generation of four CFTR transcripts with distinct 5'-UTRs, all of which encode one or more uORFs. There is increasing evidence that post-transcriptional regulation of gene expression, through modulation of mRNA stability and translation initiation, is achieved through 5'-UTR encoded elements, such as uORFs and stem-loop secondary structures (43, 48, 59). Although the cis-elements controlling post-transcriptional regulation of gene expression were first identified in yeast, similar mechanisms are now known in mammalian cells. To date over two-thirds of identified mammalian genes that encode uORFs in their 5'-UTRs are protooncogenes (60–63). However, uORFs have also been identified in a few genes with functions unrelated to cell growth control, the S-adenosylmethionine decarboxylase gene (64, 65) and Huntingtin gene (66). To these examples we now add the CFTR gene. Further, the causative mutations in the inherited diseases of familial melanoma (67, 68) and thrombocythemeia (69) create or abolish uAUGs that result in dramatic alterations in steady-state mRNA and protein levels.
It is estimated that less than 10% of eukaryotic mRNAs have an uORF in their 5'-UTR; however, very few have been investigated (48, 60). In general, uORFs lead to destabilization of the mRNA of the main ORF, secondary to disruption of ribosome scanning and reduced translation initiation at the main ORF (43). All CFTR transcripts expressed in the heart have the necessary 5'-UTR elements, uORFs with adjacent stem-loop structures, to allow post-transcriptional mechanisms to contribute to the regulation of CFTR expression. We have shown that the uORF, probably acting in concert with the adjacent stem-loop, encoded in CFTRTRAD-139 transcripts functions to reduce translation initiation efficiency at the downstream AUG of the main CFTR ORF. In addition, the uORFs encoded in exon –1C have a similar effect. With reduced translation resulting in mRNA destabilization (43), it is probable that differential stability of CFTR transcripts in the heart may be a key factor governing spatial, temporal, and pathological changes in CFTR expression. Indeed, in cardiac tissues, different levels of CFTR expression were measured over exons 1–2 compared with exons 4–6 for all CFTR transcripts, suggesting that the 5'-UTR elements involved in modulating translation efficiency from the main AUG codon may also have a role in modulating differential stability along the CFTR transcript. However, the precise control of in vivo CFTR transcript stability remains unclear.
The discovery that uORFs in CFTR 5'-UTRs modulate CFTR protein production has implications beyond the expression of CFTR in the heart, as most mouse tissues express CFTR transcripts that include an uORF (11). Also, the CFTR transcripts expressed in human fetal lung, but not adult lung, include an uORF (11), allowing the possibility that post-transcriptional mechanisms may contribute to the large changes in CFTR expression that occur during lung development. We have identified a conserved stable stem-loop structure in the 5'-UTR of traditional CFTR exon 1 that would be included in the majority of CFTR transcripts expressed in all tissues. Similar stem-loop structures have been shown to alter translation efficiency (49, 50). Whether 5'-UTR encoded secondary structures lead to widespread post-transcriptional regulation of CFTR expression is under further investigation.
The cardiac potassium channels encoded by the HERG and KvLQT1 genes are subject to alternative exon 1 usage, resulting in distinct amino-terminal protein isoforms (70). Similarly, we have shown that CFTR–1C/–1A transcripts contain an AUG codon that is in-frame with the main CFTR ORF and directs translation of an unique CFTR protein isoform, bearing a 15-amino acid truncation at the amino terminus. CFTR–1B and CFTR–1C transcripts do not encode an in-frame AUG; however, translation may initiate from downstream AUG codons in exons 3 and 4 (45). The substantially higher levels of CFTR–1C/–1A transcripts suggest that the majority of CFTR protein expressed in the heart is the truncated amino-terminal isoform.
The regulation of CFTR protein trafficking and channel activity has been shown to involve the amino terminus. Hydrophilic residues of the H3 domain of syntaxin 1A physically interact with a cytoplasmic amino-terminal helix of CFTR, encoded in exons 2 and 3 (71, 72). Furthermore, an exon 1-encoded diphenylalanine motif (Phe-16/Phe-17) regulates CFTR membrane trafficking (73). It has been suggested that loss of CFTR exon 1 would remove both motifs (55). However, although the diphenylalanine motif would be removed, the CFTR domain that interacts with syntaxin 1A would remain as it is encoded within exons 2 and 3. It was also suggested that the CFTR domain that interacts with syntaxin 1A is hydrophobic in nature and encoded at the exon 1/2 boundary (55). However, hydrophobicity analysis demonstrates that the syntaxin 1A interacting domain is in fact hydrophilic (Fig. 6B) (52). Our analysis does indeed identify a CFTR amino-terminal hydrophobic region (residues 11–26) encoded at the exon 1/2 boundary, first described by Davies (52). This hydrophobic region, truncated by loss of exon 1, has a predicted helical structure and may affect CFTR function or subcellular location by its presence or absence. The putative helical structure of the CFTR amino terminus (residues 11–26) is supported by the recent identification, from the crystal structure (4.5 Å), of an helix of similar length and location within the amino terminus (residues 10–21) of the ABC transporter homologue, MsbA, from Escherichia coli (74).
We, and others, have established an absolute correlation between CFTR mRNA expression, and the species-specific distribution of cardiac cAMP-activated chloride currents (14), present in rabbits (15, 26), guinea pigs (20, 21), and monkeys and humans (18). Antisense oligodeoxynucleotide inhibition studies have confirmed CFTR as the molecular basis of the in vivo cAMP-activated chloride current present in epithelial cells and cardiomyocytes. Primary cultures of sweat duct epithelial cells (75) and ventricular cardiomyocytes (25), as well as pancreatic duct (76) and colonic and tracheal epithelial cell lines (77), were treated with antisense oligodeoxynucleotides that bind up to 23 nucleotides surrounding the traditional CFTR exon 1 translation initiation codon, present only in the CFTRTRAD-74 and CFTRTRAD-139 transcripts. This resulted in maximal inhibition (greater than 90%) of the endogenous cAMP-activated chloride currents present in all cells of epithelial origin. In contrast, there was only a partial inhibition (40%) of the endogenous cAMP-activated chloride currents in ventricular cardiomyocytes, despite doubling the concentration of antisense oligodeoxynucleotide. In all cases, sense oligodeoxynucleotides had no effect on the cAMP-activated chloride current and neither sense nor antisense oligodeoxynucleotides had any effect on calcium-activated chloride conductance. Our finding, that CFTRTRAD-139 mRNA accounts for only a small proportion of the CFTR protein-coding transcripts present in the heart, provides an explanation for the partial inhibition of the cAMP-activated chloride current in ventricular myocytes described by Hart et al. (25). The cardiac-specific CFTR–1C/–1A transcript we have identified will not bind the antisense oligodeoxynucleotides used by Hart et al. (25), and their findings support our conclusion that CFTR–1C/–1A mRNA codes the majority of CFTR protein and cAMP-activated chloride current present in the heart. Further, it is specifically the differential expression of the CFTR–1C/–1A transcript that is absolutely correlated with differences in the whole cell current density of cAMP-activated chloride currents in epicardial versus endocardial ventricular myocytes (26), and the loss of cAMP-activated chloride conductance in cardiac hypertrophy (78).
Overall, this work identifies multiple new levels at which CFTR expression is regulated in vivo. This is the first study to show the in vivo post-transcriptional regulation of CFTR expression through modulation of translation initiation efficiency by 5'-UTR encoded elements. Furthermore, this is probably a widespread mechanism regulating CFTR expression. We are also the first to show that, through alternative exon 1 usage, a unique isoform of CFTR protein is generated and is likely to be the major CFTR isoform present in the heart. This novel curtailed form of CFTR protein, CFTR–1C/–1A, is missing functionally important amino-terminal motifs and is predicted to display unique subcellular localization and activity.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure, which shows cross-species analysis of CFTR in the human, rabbit, and murine genomes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY256886
[GenBank]
–AY256889
|| To whom correspondence should be addressed: School of Biomedical Science, University of Queensland, Brisbane, Queensland 4072, Australia. Tel.: 61-07-3365-2715; Fax: 61-07-3365-1299; E-mail: ann.trezise{at}uq.edu.au.
1 The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; LVFW, left ventricular free wall; ORF, open reading frame; uORF, upstream open reading frame; 5'-UTR, 5'-untranslated region; 5'-RACE, 5'-rapid amplification of cDNA ends; qPCR, quantitative polymerase chain reaction; eGFP, enhanced green fluorescent protein; CHO, Chinese hamster ovary; eBFP, enhanced blue fluorescent protein; uAUG, upstream AUG; gDNA, genomic DNA; LV, left ventricle; RV, right ventricle.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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