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

The sequence analysis of the human intron 2 from the Na + /Ca 2+ exchanger 1 ( NCX1 ) gene has revealed a GT repeat of variable length (10–16). The 5′ sequence of intron 2 exhibited significant homology (65–70%) with other minisatellite sequences. DNA segments at the 5′ 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′ end of the short transcripts indicated a splicing event at the intron 2/exon 2 junction (5′ site) and in the vector sequence downstream of the NCX1 insert (3′ site). Molecular dissection of the 5′‐intron 2 sequence showed that the GT repeat was required for splicing activation, whereas the remainder of the 5′‐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.