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

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

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

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

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 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