Spontaneous single nucleotide polymorphism in porcine microRNA-378 seed region leads to functional alteration

<p>Sequence variation in a microRNA (miRNA) seed region can influence its biogenesis and effects on target mRNAs; however, in mammals, few seed region mutations leading to functional alterations have been reported to date. Here, we report the identification of a single nucleotide polymorphism (SNP) with functional consequence located in the seed region of porcine miR-378. <i>In vitro</i> analysis of this rs331295049 A17G SNP showed significantly up-regulated expression of the mature miR-378 (miR-378/G). <i>In silico</i> target prediction indicated that the SNP would modulate secondary structure and result in functional loss affecting >85% of the known target genes of the wild-type miR-378 (miR-378/A), and functional gain affecting >700 new target genes, and dual-luciferase reporter assay verified this result. This report of a SNP in the seed region of miR-378 leads to functional alteration and indicates the potential for substantive functional consequences to the molecular physiology of a mammalian organism.</p> <p>The SNP changed the secondary structure of pre-miR-378 and increased the production of mutant miR-378. Functionally, the SNP lead to loss and gain of miR-378 targets.</p

A mutation in porcine pre-miR-15b alters the biogenesis of MiR-15b\16-1 cluster and strand selection of MiR-15b

<div><p>MicroRNAs (miRNAs) are small non-coding RNAs that are involved in translational regulation of the messenger RNA molecules. Sequence variations in the genes encoding miRNAs could influence their biogenesis and function. MiR-15b plays an important role in cellular proliferation, apoptosis and the cell cycle. Here, we report the identification of a C58T mutation in porcine pre-miR-15b. Through <i>in vitro</i> and <i>in vivo</i> experiments, we determined that this mutation blocks the transition from pri-miRNA to pre-miRNA, alters the strand selection between miR-15b-5p and miR-15b-3p, and obstructs biogenesis of the downstream miR-16-1. These results serve to highlight the importance of miRNA mutations and their impacts on miRNA biogenesis.</p></div

Rs334680106 blocks the processing of pri-miR-15b to pre-miR-15b.

<p>(A) Schematic diagram of the specific primers designed to amplify pri-miR-15b and pre-miR-15b; the pri-miR-15b-F1 and precursor-miR-15b-R were used to amplify pri-miR-15b, and the pri-miR-15b-F2 and precursor-miR-15b-R were used to amplify the total level of pri-miR-15b and pre-miR-15b. Quantitative RT–PCR was used to measure level of pre-miR-15b (B) and pri-miR-15b (C) in 293 cells transduced by PmR-miR-15b-W, PmR-miR-15b-M and the control PmR-mCherry expression vector. MCherry was used as the internal control. Transfection was repeated three times. Data are presented as mean ± SD. **<i>P</i><0.01.</p

Allele and genotype frequencies of pre-miR-15b in the three different pig breeds.

<p>Allele and genotype frequencies of pre-miR-15b in the three different pig breeds.</p

A mutation in porcine pre-miR-15b alters the biogenesis of MiR-15b\16-1 cluster and strand selection of MiR-15b - Fig 4

<p><b>The expression ratio of miR-15b-5p to miR-15b-3p in wild-type (A) and mutant-type (B) groups.</b> Quantitative RT–PCR was used to measure level of mature miR-15b-5p and miR-15b-3p in 293 cells transduced by PmR-miR-15b-W(A), PmR-miR-15b-M(B). U6 was used as the internal control. Transfection was repeated three times. Data are presented as mean ± SD.</p

Rs334680106 alters miRNA expression <i>in vitro</i>.

<p>(A) Schematic representation of PmR-miR-15b-wild-type (W) and PmR-miR-15b-mutant (M) overexpression vectors. Quantitative RT–PCR was used to measure level of mature miR-15b-5p(B), miR-15b-3p(C) and miR-16(D) in 293 cells transduced by PmR-miR-15b-W, PmR-miR-15b-M and the control PmR-mCherry expression vector. MCherry was used as the internal control. Transfection was repeated three times. Data are presented as mean ± SD. **<i>P</i><0.01.</p

Rs334680106 alters miRNA expression <i>in vivo</i>.

<p>Expression level of miR-15b-5p (A) and miR-15b-3p (B) in blood of the wild-type (n = 9) and mutant-type (n = 9) piglets. U6 was used as the internal control. <i>P</i>-value are indicated.</p

Additional file 4: Table S8. of A de novo silencer causes elimination of MITF-M expression and profound hearing loss in pigs

Co-segregated variants detected in re-sequencing and mutation screening. Table S9. Differential expressed genes between MITF  R/r and MITF  r/r stria vascularis (SVs). Table S10. Expression levels of melanocyte marker genes in porcine SVs. Table S11. Primer pairs used for screening the MITF gene, for qPCR and for mice genotyping. Table S12. Expression levels of SOX family members in porcine SVs. Table S13. Distribution of hearing loss phenotype and genotype in a large Rongchang pig population. (DOCX 55 kb

Additional file 1: Figure S1. of A de novo silencer causes elimination of MITF-M expression and profound hearing loss in pigs

Eye morphology defects of albino pigs. Figure S2. Three family pedigrees of mapping population. Figure S3. Images showing presence of intermediate cells in the stria vascularis of albino pigs at the embryo stage. Figure S4. Results of EMSA using probe R1 and r1. Figure S5. Genotyping of Rongchang pigs for causative mutation. Figure S6. The human orthologous of the causative mutant region found in MITF  r/r pigs are formerly lack of regulatory activity. (DOCX 6667 kb

Additional file 3: Table S5. of A de novo silencer causes elimination of MITF-M expression and profound hearing loss in pigs

SNPs detected by re-sequencing in the associated region of Rongchang pigs. Table S6. SNPs co-segregated with hearing loss phenotype in three MITF  r/r pigs and three MITF  R/R Rongchang pigs. Table S7. SNPs uniquely detected in Rongchang pigs, and homozygous in MITF  r/r . (XLSX 465 kb