Variability in porcine microRNA genes and its association with mRNA expression and lipid phenotypes

Background Mature microRNAs (miRNAs) play an important role in repressing the expression of a wide range of mRNAs. The presence of polymorphic sites in miRNA genes and their corresponding 3′UTR binding sites can disrupt canonical conserved miRNA–mRNA pairings, and thus modify gene expression patterns. However, to date such polymorphic sites in miRNA genes and their association with gene expression phenotypes and complex traits are poorly characterized in pigs. Results By analyzing whole-genome sequences from 120 pigs and wild boars from Europe and Asia, we identified 285 single nucleotide polymorphisms (SNPs) that map to miRNA loci, and 109,724 SNPs that are located in predicted 7mer-m8 miRNA binding sites within porcine 3′UTR. In porcine miRNA genes, SNP density is reduced compared with their flanking non-miRNA regions. By sequencing the genomes of five Duroc boars, we identified 12 miRNA SNPs that were subsequently genotyped in their offspring (N = 345, Lipgen population). Association analyses of miRNA SNPs with 38 lipid-related traits and hepatic and muscle microarray expression phenotypes recorded in the Lipgen population were performed. The most relevant detected association was between the genotype of the rs319154814 (G/A) SNP located in the apical loop of the ssc-miR-326 hairpin precursor and PPP1CC mRNA levels in the liver (q-value = 0.058). This result was subsequently confirmed by qPCR (P-value = 0.027). The rs319154814 (G/A) genotype was also associated with several fatty acid composition traits. Conclusions Our findings show a reduced variability of porcine miRNA genes, which is consistent with strong purifying selection, particularly in the seed region that plays a critical role in miRNA binding. Although it is generally assumed that SNPs mapping to the seed region are those with the most pronounced consequences on mRNA expression, we show that a SNP mapping to the apical region of ssc-miR-326 is significantly associated with hepatic mRNA levels of the PPP1CC gene, one of its predicted targets. Although experimental confirmation of such an interaction is reported in humans but not in pigs, this result highlights the need to further investigate the functional effects of miRNA polymorphisms that are located outside the seed region on gene expression in pigs. Supplementary Information The online version contains supplementary material available at 10.1186/s12711-021-00632-3.


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Mature microRNA transcripts (miRNAs) are short (~22 nt) non-coding RNAs   The wild ancestors of pigs were independently domesticated in the Near East In association analyses with lipid traits or gene expression phenotypes, the  inference (see previous Methods section) were used as inputs for pathway enrichment one-sided hypergeometric test of significance was applied for determining enriched 3 9 1 terms and multiple testing correction was implemented with a false discovery rate  Confirming associations between ssc-miR-326 rs319154814 genotypes and gene The hepatic mRNA levels of three of the mRNA transcripts amongst the most significantly associated with ssc-miR-326 rs319154814 (G/A) genotype were analyzed (ACTB) and TATA-Box binding protein (TBP) genes were used as endogenous were designed for each gene (Additional file 5: Measuring the expression of the ssc-miR-326 gene in pigs with different rs319154814 4 3 0 genotypes. 4 3 1 Liver samples from 9 AA and 9 GG pigs (rs319154814 genotype) were used to measure Reverse Transcription Kit (Applied Biosystems, Foster City, CA), following the 4 3 5 instructions of the manufacturer. Total RNA (10 ng) diluted in a volume of 2 μ l, was 4 3 6 used as a template to generate and pre-amplify cDNA by carrying out four consecutive 4 3 7 reactions (poly-A tailing, adaptor ligation, reverse transcription and pre-amplification).

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The resulting pre-amplified cDNA was diluted to 1:50 in Milli-Q water. To measure 4 3 9 ssc-miR-326 expression levels, both let-7a and miR-26a-5p were chosen as endogenous  and 20 AWB individuals (Additional file 1: Table S1). The distribution of these SNPs  strong genetic differentiation amongst Asian and European populations ( Fig. 2A). In contrast, domestic pigs and wild boars, and particularly those with European origin, did genetic differentiation and were more diverse than their European counterparts. With regard to miRNA variability, the 370 porcine miRNA genes annotated in   ( Table 3). For instance, the hepatic mRNA levels of the cellular FLICE-like inhibitory were amongst the most significantly associated with ssc-miR-326 genotypes ( Table 3).
The expression levels of seven of these mRNAs associated with ssc-miR-326 The mRNA levels of the PPP1CC, CFLAR and SF3A3 mRNA transcripts were SF3A3) also showed a reduced expression in pigs with AA genotype (Fig. 6A), but 6 1 7 results were not significant. We further investigated whether the ssc-miR-326 in the liver by using a specific Taqman Advanced miRNA probe assay. In this qPCR analysis, pigs with AA genotypes for the rs319154814 polymorphism showed an 6 2 1 increased ~1.9-fold expression of ssc-miR-326 transcripts compared with their GG 6 2 2 counterparts, although such difference did not reach statistical significance (P-value = 6 2 3 0.178, Fig. 6B).

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The rs333787816 (T/C) polymorphism, located in the precursor region, immediately downstream the mature ssc-miR-23a sequence, was also significantly 6 2 6 associated with 36 experimentally confirmed targeted genes in the GM muscle tissue (q-6 2 7 value < 0.1, Table 3, Additional file 14: Table S10). From these, it is worth mentioning the Argonaute RISC component 1 (AGO1) and the Argonaute RISC  Table 3). insights of putative metabolic functions of miRNAs. 6 3 5 After performing pathway enrichment analyses on the sets of putative targeted mRNA 6 3 6 transcripts significantly associated with miRNA SNP genotypes (P-value < 0.05), several biological categories were identified and reported at Additional file 17: Table   6 3 8

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To mention a few of the most relevant findings, the ssc-miR-23a genotype was 30d genotype in GM, we identified the regulation of ornithine decarboxylase activity or 6 4 5 cellular response to hypoxia, as well as mature mRNA transport after splicing 6 4 6 (Additional file 17: Table S13).

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In the liver tissue, the ssc-miR-326 genotype was significantly associated with the miR-130a genotypes (Additional file 17: Table S13). We also evaluated the association between miRNA SNPs and several lipid- related phenotypes recorded in the Lipgen population (Additional file 3: Table S3).

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More specifically, we found significant associations with myristic acid (C14:0) content 6 6 0 in both LD and GM muscles, as well as with the gadoleic acid (C20:1) content and the 6 6 1 ratio between PUFA and MUFA in the LD muscle ( association with the content of arachidic acid (C20:0). Other significant associations at 6 7 0 the nominal level were, for instance, those between the rs334590580 (T/C) SNP located 6 7 1 at the precursor stem region of ssc-miR-335 and palmitic and arachidic acids content in 6 7 2 GM tissue ( Table 4). The PCA revealed the existence of a detectable genetic differentiation between  While genetic differentiation between wild boar and pig populations was clearly 6 8 9 discernible considering the whole-genome SNP data set ( Fig. 2A), this was less evident in the PCA based on miRNA SNPs (Fig. 2B), probably because the low number (285 SNPs) of markers employed in this analysis limits the resolution with which population 6 9 2 differentiation can be detected. We have also found that the degree of population stability, localization, translation, and binding to miRNAs and RNA-binding proteins 6 9 8 [77], so in general, they are not expected to have drastic consequences on gene function 6 9 9 [77]. Purifying selection is less intense in 3'UTRs than in protein-coding regions,  We have found that, in general, miRNA loci have a substantially lowered SNP 7 0 6 density in their seeds when compared with mature and precursor regions (Fig. 4A).
When we analyzed the SNP density in miRNA loci and their flanking regions (± 1 kb), with upstream and downstream flanking sequences (Fig. 4C). These results were in  SDC1, SF3A3, FSTL1, NAA50 and ELAVL1 genes (Fig. 5), a result that was confirmed not reach statistical significance (Fig. 6A). The significant downregulation of at least in the liver tissue, we found that ssc-miR-326 log 2 relative expression is increased by 8 1 4 ~1.9 fold in pigs homozygous for the A-allele, although such result was not statistically 8 1 5 significant (P-value = 0.178, Fig. 6B).

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As previously discussed, the rs319154814 (G/A) polymorphism is located in the (G/A) that induces a steric disruption of the pri-miRNA folding structure of the hairpin, apical region can modify the efficiency with which the Drosha processing machinery, is structural consequences similar to those described for the hsa-miR-30c apical loop  One clear limitation of our experimental design is that we did not test the would be necessary to demonstrate our hypothesis that the rs319154814 (G/A) on the metabolism of myristic fatty acid has been described so far, but there are reports suggesting that several of the targets of this miRNA might be involved in carbohydrate activates acetyl-CoA carboxylase α (ACACA) and 6-phosphofructo2-kinase/fructose- factors such as sterol regulatory element-binding protein 1 (SREBF1), carbohydrate- envisaged, but this hypothesis still needs to be confirmed at the functional level. abundances in a multi-omics approach, they described three miRNA SNPs significantly  European pigs and wild boars and, in general, they display low levels of polymorphism. As expected, this reduced miRNA variability was particularly prevalent in the seed region, a finding that is likely explained by the strong effects of purifying selection 8 6 7 aiming to preserve the conservation of this critical site. We have detected one SNP in  Animal care and management procedures were carried out in accordance with national 8 7 7 guidelines for Good Experimental Practices and they were approved by the Ethical 8 7 8 Committee of the Institut de Recerca i Tecnologia Agroalimentàries (IRTA). Not applicable. Microarray expression data used in the current study were deposited in the Gene sequencing dataset from the 5 Duroc boars is available at the Sequence Read Archive as from the 5 sequenced Duroc boars are available at the following link: https://figshare.com/projects/VCF_PIGs/93140. Competing interests 8 9 6 The authors declare that they have no competing interests.
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