Role of SFRS13A in low-density lipoprotein receptor splicing

Abstract

Low-density lipoprotein receptor (LDLR) is a major apolipoprotein E (APOE) receptor and thereby is critical to cholesterol homeostasis and, possibly, Alzheimer disease (AD) development. We previously identified a single nucleotide polymorphism (SNP), rs688:C>T, that modulates LDLR exon 12 splicing and is associated with cholesterol levels in premenopausal women and with Alzheimer disease in men. To gain additional insights into LDLR splicing regulation, we seek to identify splicing factors that modulate LDLR splicing efficiency. By using an in vitro minigene study, we first found that ectopic expression of SFRS3 (SRp20), SFRS13A (SRp38), SFRS13A-2 (SRp38-2), and RBMX (hnRNP G) robustly decreased LDLR splicing efficiency. Although SFRS3 and SFRS13A specifically increased the LDLR transcript lacking exon 11, SFRS13A-2 and RBMX primarily increased the LDLR isoform lacking both exons 11 and 12. When we evaluated the relationship between the expression of these splicing factors and LDLR splicing in human brain and liver specimens, we found that overall SFRS13A expression was significantly associated with LDLR splicing efficiency in vivo. We interpret these results as suggesting that SFRS13A regulates LDLR splicing efficiency and may therefore emerge as a modulator of cholesterol homeostasis.

Figure 1. SR protein family effects on LDLR minigene splicing in vitro

HepG2 cells were co-transfected with vectors encoding different SR proteins and LDLR rs688C or rs688T in an exon 9–14 minigene. The effects of the SR proteins and rs688 allele on LDLR minigene splicing are shown as representative images (A) and quantitative results (B–E, mean ± SD, n = 3, * and + reflect p < 0.01 when compared to rs688T and rs688C minigenes, respectively, co-transfected with the negative control pEGFP vector). The faint PCR products observed between FL and Delta 11, and between Delta 12 and Delta 11+12 represent non-physiologic LDLR splice variants, i.e., FL LDLR lacking the first 74 bp of exon 14, and a Delta 13 LDLR isoform, respectively.

Figure 2. HnRNP family member effects on LDLR minigene splicing in vitro

HepG2 cells were co-transfected with vectors encoding different hnRNP family members and LDLR rs688C or rs688T-containing minigenes. The effects of the hnRNPs and rs688 allele on LDLR minigene splicing are shown as representative images (A) and quantitative results (B–E, mean ± SD, n = 3, except for HNRNPA1 and A2B1, which reflects mean ± range, n=2, * and + reflect p < 0.01 when compared to rs688T and rs688C minigenes co-transfected with the negative control pEGFP vector).

Figure 3. Splicing factors show dose-dependent effects on LDLR splicing

The indicated amounts of vectors encoding splicing factors were co-transfected with 1 μg of the vector encoding the rs688C allele LDLR minigene. The total amount of non-LDLR vector in the transfections was held constant at 1 μg by adding “negative control” pcDNA4 vector. The dose-dependent overexpression of SFRS13A, SFRS13A-2 and RBMX was confirmed by Western blots (A). Dose dependent effects on LDLR minigene splicing efficiency are shown as representative images (B) and overall quantitation (C–F, mean ± SD, n = 3).

Figure 4. SFRS13A and rs688 are associated with LDLR splicing efficiency

The relationship between LDLR splicing efficiency, SFRS13A expression and rs688 genotype are shown. As expression level of SFRS13A increased, the splicing efficiency of LDLR decreased. (A) In brain, specimens included 16 rs688C/C, 18 rs688C/T, and 19 rs688T/T. The r² for the model is 0.309. (B) In liver, specimens included 8 rs688C/C, 16 rs688C/T and 15 rs688T/T. The r² for the model is 0.213. The solid lines represent fit lines and the dashed lines represent 95% confidence intervals.