A polymorphic GT repeat from the human cardiac Na⁺Ca²⁺ exchanger intron 2 activates splicing

Abstract

The sequence analysis of the human intron 2 from the Na 1 /Ca 21 exchanger 1 (NCX1) gene has revealed a GT repeat of variable length (10±16). The 5 H sequence of intron 2 exhibited significant homology (65±70%) with other minisatellite sequences. DNA segments at the 5 H end of intron 2 were inserted in the NCX1 cDNA (3.7 kb) to reconstruct the exon 2/intron 2 junction. Transient expression of these constructs in HEK293 cells generated shortened mRNAs (< 2.5 kb). RT-PCR and ribonuclease protection analysis of the 3 H end of the short transcripts indicated a splicing event at the intron 2/exon 2 junction (5 H site) and in the vector sequence downstream of the NCX1 insert (3 H site). Molecular dissection of the 5 H -intron 2 sequence showed that the GT repeat was required for splicing activation, whereas the remainder of the 5 H -intron 2 segment was completely inactive. The results indicate that the GT repeat is a strong intronic splicing enhancer that could be involved in the regulation of NCX1 expression, possibly mediating tissue-specific alternative splicing of the mutually exclusive exons 3 and 4, and/or exon-2 circularization.Keywords: human polymorphism; intronic splicing enhancer; Na 1 Ca 21 exchanger.The plasma-membrane Na 1 /Ca 21 exchanger is a reversible transporter that couples the translocation of three Na 1 ions to one Ca 21 [1,2]. The human exchanger is composed of 944 amino acids [3,4], encoded in the 5 H region of a 7-kb mRNA [5], that is assembled by the splicing of 12 exons from the NCX1 gene, located in a 200-kb region of human chromosome 2. In addition, a 5 H -untranslated region is composed of the alternatively spliced exons 1 (a±e) [2,6]. The encoded protein (108 kDa) comprises 11 hydrophobic segments, a cleavable N-terminal signal peptide [5] and a large hydrophilic loop that includes important regulatory domains [7]. About two-thirds of the exchanger protein is encoded in an unusually large exon (exon 2, 1.8 kb), whose structure is conserved in the related NCX3 gene [6]. Tissuespecific variations in the large hydrophilic portion of the NCX1 protein is generated by alternative splicing of exons 3±7, also termed exons A±F [8]. The resulting isoforms are composed of the products of mutually exclusive exons 3 or 4 (A or B), and by different combinations of small cassettetype exons [9±12]. Alternative splicing events at the 3 H end of the canine heart 6-kb cDNA expressed in HK293 cells caused the rearrangement of the C-terminal portion of the protein, i.e. the replacement of the last C-terminal hydrophobic segments, of the last two, or the deletion of the last six hydrophobic segments [13,14]. Two main NCX1 transcripts of 7 kb and 1.8 kb have been identified in heart and in other tissues [3±5,11] corresponding to the full-length mRNA and to a circularized exon 2 form, respectively [15]. Quantitative PCR analysis on the human heart NCX1 cDNA has revealed that the most abundant mRNA was spliced immediately downstream of exon 2, apparently corresponding to the circular exon 2. It has been propos...

A threonine to isoleucine missense mutation in the pericentriolar material 1 gene is strongly associated with schizophrenia

“…36,37 There have been reports of intronic microsatellites directly effecting gene expression, such as in 97% of Freidrich's ataxia patients, and the GT microsatellite repeat from the human cardiac Na þ Ca 2 þ exchanger gene. 38,39 Microsatellites are found to be non-randomly distributed within the genomes of plants, fungi and animals, located more frequently near transcribed gene-rich areas and could provide another basis for quantitative genetic variation. [40][41][42][43] Three synonymous exonic SNPs, PCM1_11305_67, rs3780103 and rs6991775 in exons 14, 16 and 17 were observed.…”

TDP-43 Binds Heterogeneous Nuclear Ribonucleoprotein A/B through Its C-terminal Tail

“…In addition to CFTR exon 9 splicing, the isolation of TDP-43 as a highly specific (ug)m-binding protein is relevant to other genes in which (ug)m-repeated sequences have been described at the 3Ј-splice site, such as in the case of apolipoprotein AII exon 3 (31) and intron 2 of the human cardiac Na ϩ -Ca 2ϩ exchanger (32). In addition, ug-repeated sequences have been predicted to function as intronic splicing enhancer elements in the fish Fugu rubripes (33).…”

Repeat polymorphism analysis for examining genetic variability and the implications for specific traits in dog breeds

“…In case of human sirtuin 3 (SIR3) gene, a VNTR polymorphism in intron 5 has been investigated, demonstrating an allele-specific enhancer activity in vitro (Bellizzi et al, 2005). As for studies on relationship with splicing events, a polymorphic GT simple repeat in intron 2 of human cardiac Na + Ca 2+ exchanger 1 (NCX1) gene exhibits variable length, and acts as a strong intronic splicing enhancer that could regulate the NCX1 expression, possibly by controlling tissuespecific alternative splicing (Gabellini, 2001). Here, we reviewed repeat polymorphisms including microsatellites, VNTRs, and short interspersed nuclear elements (SINEs) associated with functional genes, focusing on relationship with behavioral variation and individual identification.…”