A 3'UTR polymorphism marks differential KLRG1 mRNA levels through disruption of a miR-584-5p binding site and associates with pemphigus foliaceus susceptibility

Abstract

Genetic variations mapping to 3' untranslated regions (3'UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3'UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) - an autoimmune blistering skin condition with unique endemic patterns - we investigated whether nine 3'UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene's activity through the 3'UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3'UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus.

Keywords: 3′UTR; Gene expression; KLRG1; eQTL; miR-584-5p; rs1805672.

Figure 1

Correlation plots between miR-584-5p and KLRG1 mRNA levels in PBMC of healthy donors after Log2 transformation of Cq values. Spearman correlation analysis indicated (A) significant positive correlation for the total sample (P=0.030; r_S=0.180; n=146), (B) a trend for positive correlation in carriers of the miR-584-5p-disrupting G allele (P=0.100; r_S=0.187; n=75) and (C) absence of correlation in individuals with the A/A genotype, where the miR-584-5p seed site is supposed to be fully available (P=0.495; r_S=0.086; n=65).

Figure 2

Correlation plots between miR-584-5p and SH3TC2, and KLRG1 and SH3TC2 mRNA levels in PBMC of healthy donors after Log2 transformation of Cq values. Spearman correlation analysis confirmed (A) the expected significant positive correlation between miR-584-5p and its host gene, SH3TC2, mRNA levels (P<0.0001; r_S=0.474; n=146) and revealed (B) a significant positive correlation between KLRG1 and SH3TC2 mRNA levels (P=0.002; r_S=0.250; n=146), corroborating our hypothesis that KLRG1 and SH3TC2 accumulate at directly proportional levels in PBMC, which in turn explains positive correlation between KLRG1 mRNA and miR-584-5p in the case where individuals are carriers of miR-584-5p-disrupting G allele.

Figure 3

Putative and validated interaction between miR-584-5p and the 3’UTR of KLRG1 in the context of rs1805672 polymorphism. According to TargetScan algorithm, (A) the predicted interaction of miR-584-5p with KLRG1 mRNA takes place from position 1044 to 1050 of the 3’UTR (seed sequence in bold), when an A is present at position 1044 (top), being such interaction lost when a G is present instead (bottom). Our dual-luciferase reporter assays, with Renilla as a normalizer for Firefly expression, confirmed such interaction by (B) showing that, in the presence of the A allele, luciferase activity was significantly decreased by approximately 50% in the presence of miR-584-5p (vs. “neg. control”, miRNA-negative transfection condition, left columns; P=0.006; 49.9% decreased). The mutated vector harboring the G allele showed no significant modulation of luciferase activity when co-transfected with miR-584-5p (vs. “neg. control”, miRNA-negative transfection condition, right columns; P=0.391). Comparison of both miRNA-transfected conditions confirmed the ability of the G allele to disrupt, through the 3’UTR of KLRG1, miR-584-5p-mediated luciferase downregulation (cross-hatched columns; P=0.005). Transfection of miR-584-5p mimic in PBMC resulted in (C) a decrease of approximately 65% of KLRG1 mRNA levels (vs. “neg. control”, miRNA-negative transfection condition; P=0.007; 65.3% decreased), extending the reported silencing effect of this miRNA on KLRG1 3’UTR to its full messages as well.

Figure 4

Expression Quantitative Trait Locus (eQTL) effect of rs1805672 on KLRG1. Based on RNA-Seq data from fibroblasts present in the GTEx database, we show a strong cis-eQTL effect of rs1805672 on KLRG1 mRNA levels (P=3.4×10⁻¹³; effect size=0.48). The higher KLRG1 mRNA levels in individuals with miR-584-5p-disrupting G/G genotype is noteworthy.