A Mutation In Porcine MiR-15b Precursor Alters Its Biogenesis

Micro RNAs(mi RNAs) are a class of ~22nt, single-stranded non-coding RNAs which can suppress post-transcriptional gene expression by base pairing with 3’UTR of their target m RNAs and inducing either translational repression or m RNA degradation. Mi R-15 b is a member of the mi R-15/16 family and these are playing the important role in biological processes of cell proliferation, cell apoptosis and cell cycle. In this study we identified a C/T mutation in pre-mi R-15 b of pig. To know whether the mutation could effect the biogenesis of mi R-15 b, we explored how the mutation influences the expression level of mi RNAs and their precursors through a series of in vitro and in vivo analyses. Subsequently we explored the differential expression of mi R-15 b in different organs of pig. The results are as follows:1. A C>T mutation was identified in fifty-eighth nucleotide of pre-mi R-15b(+58C>T) in Landrace and Duroc genomes and the allele frequencies of T were 0.03 and 0.36, respectively. However, this mutation was not found in Rong Chang pigs.2. The secondary structure and free-energy of wide-type and mutant-type pre-mi R-15 b was predicted by Mfold and RNAfold softwares. The results shown that the +58C>T alters the secondary structure of pre-mi R-15 b and leads its free energy decreased by 11.01%.3. We constructed wild-type(CC) and mutant-type(TT) pre-mi R-15b/mi R-16-1 expression vectors and transfected the two vectors into HEK293 cell line, respectively. Then the relative quantitative expression of mi RNAs and their precursors were assayed by Real-time quantitative PCR(q PCR). The results shown that the relative quantitative expression of mi R-15b-5p and mi R-16 in cells transfected wild-type expression vector were probably 1.6 times(very significant difference, P<0.01) that in cells transfected mutant-type expression vector and the overall relative quantitative expression of pri-mi R-15 b and pre-mi R-15 b in cells transfected wild-type expression vector was probably 1.6 times(very significant difference, P<0.01) that in cells transfected mutant-type expression vector too. The relative quantitative expression of mi R-15b-3p in cells transfected mutant-type expression vector was probably 2.3 times(very significant difference, P<0.01) that in cells transfected wild-type expression vector. The relative quantitative expression of pri-mi R-15 b in cells transfected wild-type expression vector was probably equal to cells transfected mutant-type expression vector(not significant, P>0.05). Then we found the ratio of mir-15b-5p and mir-15b-3p was 11:1 in cells transfected wild-type expression vector, but was 3.5:1 in cells transfected mutant-type expression vector.4. The expression level of mi R-15b-5p and mi R-15b-3p were detected in blood of wild-type and mutant-type Duroc pigs by q PCR. The results shown that the expression level of mi R-15b-5p in wild-type was probably twice(very significant difference, P<0.01) that in mutant-type, while the expression level of mi R-15b-3p in mutant-type was probably 5 times(very significant difference, P<0.01) that in wild-type.5. The relative quantitative expression of mi R-15b-5p in different tissues of Rongchang pigs were assayed using q PCR, the results shown that the mi R-15b-5p was highly expressed in thymus and heart and lowly expressed in liver.Our study provides a theoretical foundation for further functional studies of mi R-15 b and +58C>T and provides new ideas for functional studies of mi RNA mutation.