Verification of imprinting of two miRNAs predominantly expressed in female embryos. (A) Schematic representation of two mature miRNAs, miR-374-5p and miR-421-3p and their preceding transcript pri-miRNA. Both miRNAs were clustered and located in the intron of the Ftx (B230206F22Rik) gene on the X chromosome. Both are thought to be transcribed as pri-miRNAs and then processed to form mature miRNAs. The location of the primers used to assess the pri-miRNA and Ftx expression levels are indicated: qRT–PCR primers 207 and 208 were used for Ftx ; primers used for allelic discrimination were 057 and 058 for pri-miRNA, 116 and 117 for Ftx . The exons for Ftx are numbered according to a previous report [22] . DNA polymorphisms used for allelic discrimination are indicated with red characters. PCR primers correspond to the list in Table S3 (B) Production of mature miRNAs from pre-miRNAs in female blastocysts. Although two mature forms of miRNA, miR-374-5p/miR-374-3p and miR-421-5p/miR-421-3p are registered in miRBase, only miR-374-5p and miR-421-3p were detected at the blastocyst stage. Orange coloring shows mismatch sequences compared with the reference sequences, suggesting miRNA editing or misreading by the 454 sequencer. (C) Expression of pri-miRNA in wild-type male and female blastocysts obtained from the uterus. Female-predominant expression was observed as well as previously reported X-linked imprinted genes such as Xist and Rhox5 . (D) Verification of imprinting of the pri-miRNA for miR-374-5p and miR-421-3p in preimplantation mouse embryos. Pri-miRNA was expressed from the paternal B6 allele in JF1× C57BL/6 crosses and from the paternal JF1 allele in the reciprocal B6× (JF1× B6) cross. The arrows indicate the polymorphism sites that were used in this assay.
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