MicroRNA transcriptome in the newborn mouse ovaries determined by massive parallel sequencing

Abstract

Small non-coding RNAs, such as microRNAs (miRNAs), are involved in diverse biological processes including organ development and tissue differentiation. Global disruption of miRNA biogenesis in Dicer knockout mice disrupts early embryogenesis and primordial germ cell formation. However, the role of miRNAs in early folliculogenesis is poorly understood. In order to identify a full transcriptome set of small RNAs expressed in the newborn (NB) ovary, we extracted small RNA fraction from mouse NB ovary tissues and subjected it to massive parallel sequencing using the Genome Analyzer from Illumina. Massive sequencing produced 4 655 992 reads of 33 bp each representing a total of 154 Mbp of sequence data. The Pash alignment algorithm mapped 50.13% of the reads to the mouse genome. Sequence reads were clustered based on overlapping mapping coordinates and intersected with known miRNAs, small nucleolar RNAs (snoRNAs), piwi-interacting RNA (piRNA) clusters and repetitive genomic regions; 25.2% of the reads mapped to known miRNAs, 25.5% to genomic repeats, 3.5% to piRNAs and 0.18% to snoRNAs. Three hundred and ninety-eight known miRNA species were among the sequenced small RNAs, and 118 isomiR sequences that are not in the miRBase database. Let-7 family was the most abundantly expressed miRNA, and mmu-mir-672, mmu-mir-322, mmu-mir-503 and mmu-mir-465 families are the most abundant X-linked miRNA detected. X-linked mmu-mir-503, mmu-mir-672 and mmu-mir-465 family showed preferential expression in testes and ovaries. We also identified four novel miRNAs that are preferentially expressed in gonads. Gonadal selective miRNAs may play important roles in ovarian development, folliculogenesis and female fertility.

Figure 1

Pie chart of (A) sequences that mapped to known RNAs, repeats and genes and (B) genomic context of sequences mapped to known miRNAs. Sequences were clustered based on overlapping mapping coordinates and intersected with known miRNAs, snoRNAs, piRNA clusters and repeats. scRNA, small cytoplasmic RNA; srpRNA, signal recognition particle RNA; SINE, short interspersed repetitive elements; LINE, long interspersed repetitive elements.

Figure 2

Chromosomal location of miRNAs, piRNAs and snoRNAs based on the number of sequence reads (A) and number of genes (B).

Figure 3

Multi-tissue RT–PCR of selected known miRNAs. Small RNA fraction was isolated and cDNA synthesized from 11 different mouse tissues followed by PCR. Expression profiles of (A) mmu-mir-202, mmu-mir-503 and mmu-mir-672 and (B) mmu-mir-465 cluster (mmu-mir-465a, mmu-mir-465b, mmu-mir-465c and mmu-mir-465-3p) are shown. U6 RNA amplification was used as a positive control.

Figure 4

Multi-tissue RT–PCR on selected novel miRNAs and isomiRs. Small RNA rich cDNA library was generated from 11 different mouse tissues and PCR was performed. Expression profiles of miRNAs preferentially expressed in the gonads (A) is shown (novel 11, novel 12, novel 18, novel 20, and novel 23), and (B) isomiRs (mir-135a-2-3p and mmumir-871-3p) are shown here. NovelRNA 361 belongs to a group of RNA species that were not predicted to derive from pre-miRNA. U6 was used as a positive control.