Smoking exposure alters splicing of the nicotinic acetylcholine receptor subunit CHRNA5

Cigarette smoking is the major preventable cancer risk factor. The missense variant rs16969968 (G > A, D398N) in exon 5 of CHRNA5 on chromosome 15q25.1 alters activity of the nicotinic acetylcholine receptor (α5-nAChR) and is associated with increased smoking intensity and cancer risk. We hypothesized that smoking exposure might affect alternative splicing of CHRNA5, which is mainly limited to exon 5, and thereby modulate genetic associations reported for rs16969968.

In human tissues and cell lines, we detected five main alternative CHRNA5 isoforms, each lacking 130-786 bp of exon 5, including the region with rs16969968. In tumors from The Cancer Genome Atlas (TCGA), only 52.4% of all exon 5 splice junctions corresponded to the full-length isoform. We modeled cigarette smoking exposure by treating cell lines representing smoking-related cancers - A549 (lung) and UMUC3 (bladder) - with cigarette smoke concentrate (CSC). Splicing of CHRNA5-exon 5 was evaluated in the context of a transiently expressed Exontrap-CHRNA5-exon5 minigene using long-read targeted cDNA sequencing and RT-qPCR. Long-term, but not short-term, smoking exposure significantly altered the relative abundance of several CHRNA5 splice isoforms, but independently of rs16969968 alleles.

Smoking exposure can modulate CHRNA5 splicing and, consequently, the composition and function of α5-nAChR, the receptor regulating the response to smoking. Thus, smoking-induced alterations of CHRNA5-exon 5 splicing can influence nicotine dependence and cancer risk, acting both independently of and complementary to the genetic risk conferred by the rs16969968-A variant.