Functional sQTLs regulating PTK2B exon 31 splicing uncover an RNA-dependent modulation of its kinase activity and cellular phenotype

Genome-wide association studies have shown that polymorphisms in PTK2B (Protein Tyrosine Kinase 2 Beta), encoding the calcium-sensitive tyrosine kinase PYK2 (Proline-rich tyrosine kinase 2), are associated with an increased risk of Alzheimer’s disease (AD). However, it remains unclear which genetic variants of PTK2B are functionally important. A recent transcriptome-wide association study has identified alternative splicing of exon 31 in PTK2B as being associated with AD, and proposed rs2251430 as the relevant splicing quantitative trait locus (sQTL). Here, we aimed to elucidate the regulatory mechanisms and functional significance of exon 31 splicing. Alternative splicing of exon 31 was evaluated using publicly available RNA-seq data. To investigate the regulation of exon 31 splicing, we performed RT-PCR, RNA pull-down assays, Liquid Chromatography–Tandem Mass Spectrometry, and RNA immunoprecipitation. Western blotting, immunofluorescence, and co-immunoprecipitation analyses were conducted to examine the effects of exon 31 and its polymorphisms on PYK2 protein expression and function. The impact of exon 31 on cellular phenotypes was further assessed using antisense oligonucleotide (ASO) treatment in HeLa and HMC3 cells. We detected exon 31 skipping in certain human cell types, but not in mice, and observed its increase in microglia during AD progression. We then identified rs751019 and rs751018, both located near rs2251430, as sQTLs that affect exon 31 splicing, with rs751019 having a greater impact. Among RNA-binding proteins that interact with the surrounding sequence of rs751019, PCBP1 and PCBP2 were found to redundantly regulate exon 31 inclusion. PYK2 lacking exon 31 exhibited reduced protein expression and kinase activity, which in turn resulted in diminished phosphorylation of its substrate, GSK3β, with little change in calmodulin binding and intracellular localization. Moreover, ASO-induced exon 31 skipping decreased cell migration in a wound healing assay. It also increased multinucleation in human microglia-like HMC3 cells, a process recently identified in protective microglia. Our study validates rs751019 and rs751018 as functional sQTLs that influence PTK2B exon 31 splicing. It also identifies species-specific alternative splicing of exon 31 as a modulator of PYK2 expression and kinase activity, thereby linking sQTL-associated genetic variants to molecular and cellular changes that may underlie AD pathogenesis.