Complexity of the 5'UTR region of the CLCN5 gene: eleven 5'UTR ends are differentially expressed in the human kidney

Abstract

Background: Dent disease 1 represents a hereditary disorder of renal tubular epithelial function associated with mutations in the CLCN5 gene that encoded the ClC-5 Cl-/H+ antiporter. All of the reported disease-causing mutations are localized in the coding region except for one recently identified in the 5'UTR region of a single patient. This finding highlighted the possible role for genetic variability in this region in the pathogenesis of Dent disease 1.The structural complexity of the CLCN5 5'UTR region has not yet been fully characterized. To date 6 different 5' alternatively used exons--1a, 1b, 1b1 and I-IV with an alternatively spliced exon II (IIa, IIb)--have been described, but their significance and differential expression in the human kidney have not been investigated. Therefore our aim was to better characterize the CLCN5 5'UTR region in the human kidney and other tissues.

Methods: To clone more of the 5' end portion of the human CLCN5 cDNA, total human kidney RNA was utilized as template and RNA ligase-mediated rapid amplification of cDNA 5' ends was applied.The expression of the different CLCN5 isoforms was studied in the kidney, leucocytes and in different tissues by quantitative comparative RT/PCR and Real--Time RT/PCR.

Results: Eleven transcripts initiating at 3 different nucleotide positions having 3 distinct promoters of varying strength were identified. Previously identified 5'UTR isoforms were confirmed, but their ends were extended. Six additional 5'UTR ends characterized by the presence of new untranslated exons (c, V and VI) were also identified. Exon c originates exon c.1 by alternative splicing. The kidney uniquely expresses all isoforms, and the isoform containing exon c appears kidney specific. The most abundant isoforms contain exon 1a, exon IIa and exons 1b1 and c. ORF analysis predicts that all isoforms except 3 encode for the canonical 746 amino acid ClC-5 protein.

Conclusions: Our results confirm the structural complexity of the CLCN5 5'UTR region. Characterization of this crucial region could allow a clear genetic classification of a greater number of Dent disease patients, but also provide the basis for highlighting some as yet unexplored functions of the ClC-5 proton exchanger.

Figure 1

Characterization of the CLCN5 5′UTR variant 4 and alternative variant 4 by RACE PCR and sequencing. + 1 indicates the putative transcriptional start site of alternative variant 4, located at intron 1a, 1001 nt upstream of the first nucleotide of exon 1b described by Fisher et al. (2). For variant 4 it was not been possible to obtain the full length cDNA but probably the two variants have the same transcription start site and differ only regarding the absence/presence of intron 1b. Coloured boxes represent exons and open boxes represent introns.

Figure 2

Characterization of the CLCN5 5′UTR variants 6 and 7 by RACE PCR and sequencing. + 1, which is located at intron 1a, indicates the putative mRNA variant 7 transcriptional start site that is the same of alternative variant 4. Exon c of 287 bp after 5’ alternative splicing originates two different mRNAs containing an exon c.1 of 102 bp (variant 6) and exon c of 287 bp (variant 7). Coloured boxes represent exons and open boxes represent introns.

Figure 3

Characterization of the new CLCN5 5′UTR long transcripts by RACE PCR and sequencing. The four long transcripts contain the already described exons I-IV and the new exons V and VI (variants 8-11) (131 bp and 194 bp long, respectively), located 7820 bp and 11977 bp downstream of the exon IV respectively . + 1 indicates the putative transcriptional start site located at nucleotide - 660 in respect to the ATG initiation codon in exon III. Coloured boxes represent exons and open boxes represent introns.

Figure 4

Genomic organization of CLCN5 5′UTR region. The CLCN5 5’ UTR region consists of 8 different 5’ alternatively used exons, some of this, remains untranslated. As a result of 5’ alternative splicing (represented by two vertical bars) in exons II and 1b, 11 different mRNAs are generated. Transcription initiates from three different start sites (represented by an arrow) and there are three translation start sites (represented by an inverted triangle). Coloured boxes represent exons and the connecting lines between boxes the introns.

Figure 5

Real time PCR quantification of CLCN5 isoforms in the human kidney. Data were normalized to GAPDH housekeeping gene; the isoforms were quantified using the mRNA variant 3, the most abundantly expressed, as a reference value (10^0). The y- axis reports the expression values in logarithmic scale.