Schematic representation of the imprinted mouse distal 12 domain (human 14q32). The position of several imprinted genes is indicated by squares (Gtl2 and protein-coding genes), vertical bars (snoRNA genes), or triangles (miRNA genes). Maternally expressed and paternally expressed genes are filled in pink and blue, respectively. Biallelically expressed genes and genes with undetermined imprinted status are colored in black and grey, respectively. Open triangles represent in silico-predicted miRNA gene that we failed to experimentally detect (i.e., pre-mir-L, pre-mir-H, pre-mir-G, pre-mir-I, pre-mir-Q). Two recently described miRNA genes—mir-342 and mir-345 (Kim et al. 2004)—map within an intron of Ev1 and between Yy1 and Wars genes, respectively. Imprinting status of mir-342 remains unsolved as we failed to detect it in embryos. An intergenic germ-line derived differentially methylated region (IG-DMR) located between Dlk1 and Gtl2 genes is represented by circles (filled indicates hypermethylated; open, hypomethylated). Mat indicates maternal chromosome; Pat, paternal chromosome. The figure is not drawn to scale.

Multiple sequence alignment of the three human miRNA families. The names of the pre-miRNA variants belonging to the A-, B-, and C-types are indicated at the left in A, B, and C, respectively. Conservation of each nucleotide position is denoted by shading according to GeneDoc software: black (100%), dark gray (80% to 99%), and clear gray (60% to 79%). Sequence alignment of the conserved murine pre-miRNAs belonging to the A-, B-, and C-types is shown in Supplemental data S2.

Detection of large transcripts overlapping several miRNA genes. Ten micrograms of DNAse I-, proteinase K-treated total RNAs extracted from the head of mouse embryo (E17.5) was subjected to RT-PCR with appropriate primers (numbered arrows) mapping outside from the ∼70-nt pre-miRNA genes. The location of the primers and expected sizes of the amplification products are shown on the top part of the figure (not drawn to scale). Primer sequences are available on request. RT indicates reverse transcriptase (Superscript II, Invitrogen).

Identification of computationally predicted miRNA genes at the human imprinted 14q32 domain. (A) Distribution of the conserved hairpins between human and mouse along the 1-Mb-long imprinted 14q32 domain. Vertical bars indicate the number of in silico-predicted hairpins found within each 50-kb-long region. Blue bars indicate the number of human hairpins at the 14q32 domain; red bars, the number of conserved hairpins between mouse and human; and yellow bars, the number of conserved hairpins giving rise to a miRscan score >10 (Lim et al. 2003). The relative positions of the overlapping human BACs covering the analyzed domain are indicated below the histogram. (B) A large cluster of miRNA genes mapping downstream from the C/D snoRNA gene cluster. miRNA genes belonging to the A-, B-, and C-types are represented by green, red, and blue arrows, respectively, and miRNAs encoded by a single-copy gene (miR-D, G, H, I, K) are indicated as black arrows. Note that four miRNA genes—miR-A22, miR-K, miR-A23, and miRA24—are embedded within a conserved CpG island (indicated by a gray rectangle) from which the most conserved sequences lie within the pre-miRNA genes (data not shown). Dotted lines denote splicing events identified by ESTs analysis. Numbering indicates the relative position of the miRNA gene cluster (in nucleotides) within the BAC AL132709 sequence. The picture is drawn to scale.

Expression of in silico-predicted miRNA genes. Tissue-specific (A) and developmental gene expression pattern (B) of the miRNAs analyzed by primer extension assay. H indicates head; T, trunk. 5.8S and let-7 probes have been used as gel loading controls (Northern blot analysis). (C) The miRNA genes are only expressed from the maternally inherited chromosome. Ten micrograms of total RNA extracted from wild-type embryos, embryos with maternal uniparental disomy for chromosome 12 (matUPD12), or embryos with paternal uniparental disomy for chromosome 12 (patUPD12) have been subjected to primer extension assay. (D) The miRNA imprinted gene expression is controlled by the intergenic germline-derived differentially methylated region (IG-DMR). Ten micrograms of total RNA extracted from embryo with a deletion of the IG-DMR from either the maternally or the paternally inherited chromosomes (as specified at the top of the picture) have been subjected to primer extension analysis.