Genome-wide screening for genetic variants in polyadenylation signal (PAS) sites in mouse selection lines for fatness and leanness

Abstract

Alternative polyadenylation (APA) determines mRNA stability, localisation, translation and protein function. Several diseases, including obesity, have been linked to APA. Studies have shown that single nucleotide polymorphisms in polyadenylation signals (PAS-SNPs) can influence APA and affect phenotype and disease susceptibility. However, these studies focussed on associations between single PAS-SNP alleles with very large effects and phenotype. Therefore, we performed a genome-wide screening for PAS-SNPs in the polygenic mouse selection lines for fatness and leanness by whole-genome sequencing. The genetic variants identified in the two lines were overlapped with locations of PAS sites obtained from the PolyASite 2.0 database. Expression data for selected genes were extracted from the microarray expression experiment performed on multiple tissue samples. In total, 682 PAS-SNPs were identified within 583 genes involved in various biological processes, including transport, protein modifications and degradation, cell adhesion and immune response. Moreover, 63 of the 583 orthologous genes in human have been previously associated with human diseases, such as nervous system and physical disorders, and immune, endocrine, and metabolic diseases. In both lines, PAS-SNPs have also been identified in genes broadly involved in APA, such as Polr2c, Eif3e and Ints11. Five PAS-SNPs within 5 genes (Car, Col4a1, Itga7, Lat, Nmnat1) were prioritised as potential functional variants and could contribute to the phenotypic disparity between the two selection lines. The developed PAS-SNPs catalogue presents a key resource for planning functional studies to uncover the role of PAS-SNPs in APA, disease susceptibility and fat deposition.

Fig. 1

The number of publications extracted from PubMed using keywords related to polyadenylation, PAS-SNPs and their relationship with diseases and obesity, and those found by manual screening. The used keywords are summarized in Supplementary Table S1

Fig. 2

Workflow and main results. Steps include Genotyping of Fat and Lean mouse selection lines, identification of PAS-SNPs, GO enrichment analysis by MonaGO, identification of disease-related genes (MGI) and construction of gene-disease interaction network visualized by Cytoscape, identification of common (genes containing PAS-SNPs in both lines), and obesity and APA-related genes having PAS-SNPs identified in Fat or Lean line

Fig. 3

Multiple-species alignment of parts of the selected genes with PAS-SNPs with marked genetic variants, PAS motifs, and APA sites. Legend: capital red letters—nucleotides in 3′ UTR, capital black letters shaded light blue—3′ UTR variants, small green letters—nucleotides downstream of the gene, small black letters shaded grey—downstream variants, small grey letters—nucleotides in the intron, small white letters shaded blue—intronic variants

Fig. 4

Disease network of genes with PAS-SNPs identified in Fat A and Lean B mouse selection lines for body fat percentage

Fig. 5

PAS-SNPs within Smyd3. ↑—altered PAS hexamer to a more used PAS, ↓—altered PAS hexamer to a more used PAS, X—lost PAS by SNP. Red—Fat line, Green—Lean line. Approximate miRNA target locations are also indicated (miRTarBase—predicted by miRanda)

Fig. 6

Visual identification of potentially functional PAS-SNPs. A Location of PAS-SNPs, Affymetrix probes and their expressions within Nmnat1. 3rd track, black rectangles—Affymetrix probes with no expression difference between the lines, red rectangles – the expression being higher in the Fat line compared to the Lean line, red vertical line denotes PAS-SNPs identified in the Fat line. B Location of SNPs, PAS motifs and APA sites. Legend: coloured 6 bp hexamers—PAS motifs (the colour denotes their abundance in mouse genome), bigger underlined nucleotides in bold—locations of SNPs, nucleotides coloured black and written white—APA site ID (most often cleavage site in this region according to PolyASite 2.0 portal), nucleotides in a black box—APA cluster (bigger the box more variable the cleavage site)