A natural “GA” insertion mutation in the sequence encoding the 3′UTR of CXCL12/SDF-1α: Identification, characterization, and functional impact on mRNA splicing

The CXCL12 gene produces a series of transcript variants through alternative splicing at the 3′ end of its pre-mRNA. This study explores the biological activities of these alternative transcripts and the mechanisms involved in the regulation of CXCL12 transcription and RNA splicing. We identified a “GA” insertion mutation in the region of CXCL12α DNA encoding the conserved 3′UTR. This variant transcript was named CXCL12-3′GA+. The mutation occurred at a frequency of 13.2% in healthy Chinese individuals. However, its frequency in healthy Caucasians was 22.6%, significantly higher than what was observed in the Chinese. Genomic analysis indicated that the GA+ mutation likely encodes a G-quadruplex structure in close proximity to a cluster of important AU-rich elements (AREs) that are well-established regulators of mRNA stability at the 3′UTR. Experiments using molecular constructs encoding the 3′UTR of CXCL12 revealed that the GA+ allele can significantly increase gene expression compared to the WT allele. Further studies uncovered that the WT allele was associated with the production of a 225-bp minor transcript isoform (MTI) through alternative splicing resulting in the deletion of exon 2. ARMS-PCR using samples collected from cultured PBMCs of WT/GA+ genotype carriers indicated that the GA+ allele was preferentially transcribed compared to the WT allele. In summary, the study demonstrates that a GA insertion in the region encoding the 3′UTR of CXCL12α may affect gene expression through alternative mRNA splicing. This finding provides a basis for understanding how multiple elements in the sequence encoding the 3′UTR of the CXCL12 gene regulates its transcription and may lead to insights about diseases involving abnormal CXCL12α expression.