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. 2017 May 19;45(9):5387-5398.
doi: 10.1093/nar/gkx027.

Roles of MIWI, MILI and PLD6 in small RNA regulation in mouse growing oocytes

Yuka Kabayama  1   2 , Hidehiro Toh  1 , Ami Katanaya  3 , Takayuki Sakurai  4 , Shinichiro Chuma  3 , Satomi Kuramochi-Miyagawa  5 , Yumiko Saga  4 , Toru Nakano  5 , Hiroyuki Sasaki  1
Affiliations

Roles of MIWI, MILI and PLD6 in small RNA regulation in mouse growing oocytes

Yuka Kabayama et al. Nucleic Acids Res. .

Abstract

The mouse PIWI-interacting RNA (piRNA) pathway produces a class of 26-30-nucleotide (nt) small RNAs and is essential for spermatogenesis and retrotransposon repression. In oocytes, however, its regulation and function are poorly understood. In the present study, we investigated the consequences of loss of piRNA-pathway components in growing oocytes. When MILI (or PIWIL2), a PIWI family member, was depleted by gene knockout, almost all piRNAs disappeared. This severe loss of piRNA was accompanied by an increase in transcripts derived from specific retrotransposons, especially IAPs. MIWI (or PIWIL1) depletion had a smaller effect. In oocytes lacking PLD6 (or ZUCCHINI or MITOPLD), a mitochondrial nuclease/phospholipase involved in piRNA biogenesis in male germ cells, the piRNA level was decreased to 50% compared to wild-type, a phenotype much milder than that in males. Since PLD6 is essential for the creation of the 5΄ ends of primary piRNAs in males, the presence of mature piRNA in PLD6-depleted oocytes suggests the presence of compensating enzymes. Furthermore, we identified novel 21-23-nt small RNAs, termed spiRNAs, possessing a 10-nt complementarity with piRNAs, which were produced dependent on MILI and independent of DICER. Our study revealed the differences in the biogenesis and function of the piRNA pathway between sexes.

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Figures

Figure 1.
Figure 1.
Expression of piRNA-related genes in wild-type oocytes and nuage formation in Mili and Pld6 mutant oocytes. (A) Expression of the genes involved in the piRNA biogenesis/function. Growing oocytes were obtained from P10 and P20 ovaries and fully grown oocytes (FGOs) from adult ovaries. RT-PCR was performed with total RNA from the indicated tissues and cells from wild-type mice. Gapdh was used as a control. (B) Expression of the above genes in oocytes was studied using published RNA sequencing data (accession no. GSE70116) (56). FPKM, Fragments per kilobase of exon per million mapped reads; NGO, non-growing oocyte (10–40 μm); GO1, growing oocyte (25–70 μm); GO2, growing oocyte (50–70 μm); FGO, fully grown oocyte (>70 μm). (C) Expression of the above proteins in ovaries. Western blot analysis was performed with total proteins from ovaries and testes at indicated stages. (D) Electron microscopy of Mili−/− and Pld6−/− oocytes in P14 ovary sections. The control sections were from either a wild-type or a heterozygous ovary. The arrows indicate the nuage structure formed in the inter-mitochondrial spaces. The scale bars are 500 nm.
Figure 2.
Figure 2.
piRNAs profiles in wild-type and mutant oocytes. (A) Nucleotide compositions at the first and tenth positions of 21–23-nt RNAs from the 95 putative endo-siRNA clusters and 26–30-nt RNAs from the 250 putative piRNA clusters in wild-type ovaries. (B) Nucleotide compositions at the 1st and 10th positions of 21–23-nt (putative endo-siRNAs) and 26–30-nt RNAs (putative piRNAs) derived from retrotransposons in wild-type ovaries. (C) Relative frequencies of the lengths of complementarity detected between the 5΄ portions of L1-derived 26–30-nt RNAs (putative piRNAs) and between those of intracisternal A-particle (IAP)-derived 26–30-nt RNAs in wild-type ovaries. Small RNA reads matching the consensus sequences of L1 (L1MdTf_I) and IAP (MIA14) with up to one mismatch were used. (D) Size distributions of the cluster- and retrotransposon-derived piRNAs in wild-type (WT), Mili−/−, Miwi−/− and Pld6−/− ovaries at P20. The read number was normalized by the miRNA count in each sample. (E) Relative piRNA expression from individual retrotransposon families in Mili−/− and Pld6−/− ovaries.
Figure 3.
Figure 3.
Role of piRNAs in retrotransposon repression in oocytes. MA plots of expression of retrotransposon-derived RNAs in Mili−/− and Pld6−/− ovaries at P20. Each spot indicates the mean (x-axis) and fold change (y-axis) of a particular retrotransposon in three biological replicates. Only those showing reads per kilobase of exons per million mapped reads (RPKM) > 50 in wild-type ovaries were colored. The dashed lines indicate twice or half of the expression level in wild-type ovaries.
Figure 4.
Figure 4.
A class of 21–23-nt RNAs produced via the piRNA pathway, not via the endo-siRNA pathway. (A) Relative expression of 21–23-nt small RNAs derived from individual retrotransposon families in Mili−/− and Pld6−/− ovaries. (B) Relative frequencies of complementarity lengths between the 5΄ portions of L1-derived 21–23-nt RNAs and those of 26–30-nt piRNAs in wild-type ovaries at P20. Small RNA reads matching the L1MdTf_I consensus sequences were selected, allowing for one mismatch and then used. (C) Nucleotide compositions at the first and tenth positions of L1-derived 21–23-nt RNAs showing 10-nt complementarity with L1-derived 26–30-nt piRNAs. Small RNA reads matching the L1MdTf_I consensus sequences were selected, allowing for one mismatch and then used. (D) Size distributions of cluster-derived (left) and retrotransposon-derived (right) small RNAs expressed in control (Dicerflox/+, Zp3-cre+) and DicerCKO ovaries. The read number was normalized by the miRNA count in each sample. (E) Relative frequencies of complementarity lengths between the 5΄ portions of L1-derived 21–23-nt RNAs and those of 26–30-nt piRNAs expressed in P20 DicerCKO ovaries. Small RNA reads matching the L1MdTf_I consensus sequences with up to one mismatch were used. (F) Size distributions of L1-derived small RNAs bound to MILI protein in P10 ovaries. (G) Relative frequencies of complementarity lengths between the 5΄ portions of L1-derived 21–23-nt RNAs and those of 26–30-nt piRNAs bound to MILI protein. Small RNA reads matching the L1MdTf_I consensus sequence with up to one mismatch were used. (H) Nucleotide compositions at the 1st and 10th positions of L1-derived 21–23-nt RNAs bound to MILI protein.
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