ADAD2 functions in spermiogenesis and piRNA biogenesis in mice.

BACKGROUND: Adenosine deaminase domain containing 2 (ADAD2) is a testis-specific protein composed of a double-stranded RNA binding domain and a non-catalytic adenosine deaminase domain. A recent study showed that ADAD2 is indispensable for the male reproduction in mice. However, the detailed functions of ADAD2 remain elusive. OBJECTIVES: This study aimed to investigate the cause of male sterility in Adad2 mutant mice and to understand the molecular functions of ADAD2. MATERIALS AND METHODS: Adad2 homozygous mutant mouse lines, Adad2-/- and Adad2Δ/Δ , were generated by CRISPR/Cas9. Western blotting and immunohistochemistry were used to reveal the expression and subcellular localization of ADAD2. Co-immunoprecipitation tandem mass spectrometry was employed to determine the ADAD2-interacting proteins in mouse testes. RNA-sequencing analyses were carried out to analyze the transcriptome and PIWI-interacting RNA (piRNA) populations in wildtype and Adad2 mutant testes. RESULTS: Adad2-/- and Adad2Δ/Δ mice exhibit male-specific sterility because of abnormal spermiogenesis. ADAD2 interacts with multiple RNA-binding proteins involved in piRNA biogenesis, including MILI, MIWI, RNF17, and YTHDC2. ADAD2 co-localizes and forms novel granules with RNF17 in spermatocytes. Ablation of ADAD2 impairs the formation of RNF17 granules, decreases the number of cluster-derived pachytene piRNAs, and increases expression of ping-pong-derived piRNAs. DISCUSSION AND CONCLUSION: In collaboration with RNF17 and other RNA-binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.

Effects of transgene insertion loci and copy number on Dnmt3L gene silencing through antisense transgene-derived PIWI-interacting RNAs.

PIWI-interacting RNAs (piRNAs), which are germ cell-specific small RNAs, are essential for spermatogenesis. In fetal mouse germ cells, piRNAs are synthesized from sense and antisense RNAs of transposable element sequences for retrotransposon silencing. In a previous study, we reported that transgenic mice expressing antisense-Dnmt3L under the control of the Miwi2 promoter (Tg-Miwi2P-asDnmt3L) exhibited piRNA-mediated DNMT3L down-regulation. In this study, two transgene integration loci (B3 and E1) were identified on chromosome 18 of the Tg-Miwi2P-asDnmt3L mice; these loci were weak piRNA clusters. Crossbreeding was performed to obtain mice with the transgene cassette inserted into a single locus. DNMT3L was silenced and spermatogenesis was severely impaired in mice with the transgene cassette inserted at the B3 locus (Tg-B mice). In contrast, spermatogenesis in mice bearing the transgene at the E1 locus (Tg-E mice) was normal. The number of piRNAs for Dnmt3L in Tg-B mice was eightfold higher than that in Tg-E mice. Therefore, both gene silencing and impaired spermatogenesis depended on the transgene copy number rather than on the insertion loci. Additionally, the endogenous Dnmt3L promoter was not methylated in Tg mice, suggesting that Dnmt3L silencing was caused by post-transcriptional gene silencing. Based on these data, we discuss a piRNA-dependent gene silencing mechanism against novel gene insertions.

MORC3, a novel MIWI2 association partner, as an epigenetic regulator of piRNA dependent transposon silencing in male germ cells.

The PIWI (P-element-induced wimpy testis)-interacting-RNA (piRNA) pathway plays a crucial role in the repression of TE (transposable element) expression via de novo DNA methylation in mouse embryonic male germ cells. Various proteins, including MIWI2 are involved in the process. TE silencing is ensured by piRNA-guided MIWI2 that recruits some effector proteins of the DNA methylation machinery to TE regions. However, the molecular mechanism underlying the methylation is complex and has not been fully elucidated. Here, we identified MORC3 as a novel associating partner of MIWI2 and also a nuclear effector of retrotransposon silencing via piRNA-dependent de novo DNA methylation in embryonic testis. Moreover, we show that MORC3 is important for transcription of piRNA precursors and subsequently affects piRNA production. Thus, we provide the first mechanistic insights into the role of this effector protein in the first stage of piRNA biogenesis in embryonic TE silencing mechanism.

GPAT2 is required for piRNA biogenesis, transposon silencing, and maintenance of spermatogonia in mice†.

PIWI-interacting RNAs (piRNAs), a subclass of germ cell-specific noncoding small RNAs, are essential for de novo DNA methylation of retrotransposon genes in embryonic testes. PIWIL2/MILI, one of three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably via piRNA, and normal spermatogenesis. In vitro analysis using germline stem cells (GS cells) revealed that glycerol-3-phosphate acyltransferase 2 (GPAT2), which is a mitochondrial outer membrane protein involved in generation of lysophosphatidic acid (LPA) and highly expressed in testes, plays important roles in spermatogenesis. Namely, GPAT2 binds to PIWIL2 and is closely involved in the biogenesis of piRNAs; this process is independent of its enzymatic activity on LPA. However, GS cells recapitulate only a limited phase of spermatogenesis and the biological functions of GPAT2 remain largely unknown. In this study, we generated GPAT2-deficient mice and conducted comprehensive analyses. The deficient mice showed defective piRNA production and subsequent de-silencing of IAP and Line-1 retrotransposons in fetal testes. In addition, apoptosis of pachytene spermatocytes was observed. These abnormalities were all common to the phenotype of PIWIL2-deficient mice, in which piRNA production was impaired. GPAT2-deficient mice exhibited apoptosis in spermatogonia at the neonatal stage, which was not observed in PIWIL2-deficient mice. These data show that GPAT2 plays a critical role in preventing apoptosis in spermatogonia.

Relationship between PIWIL4-Mediated H3K4me2 Demethylation and piRNA-Dependent DNA Methylation.

Retrotransposon genes are silenced by DNA methylation because of potential harm due to insertional mutagenesis. DNA methylation of retrotransposon genes is erased and re-established during male germ cell development. Both piRNA-dependent and piRNA-independent mechanisms are active during the re-establishment process, with the piRNA-independent mechanism occurring first. In this study, we analyzed the role of PIWIL4/MIWI2 in the modification of histone H3 and subsequent piRNA-dependent DNA methylation. Dimethylation at H3K4 is highly enriched at piRNA-dependent methylated regions and anti-correlated with de novo DNA methylation during the phase of piRNA-independent DNA methylation. In addition, PIWIL4, which binds the H3K4 demethylases KDM1A and KDM5B, is required for removing H3K4me2 marks. These data show that PIWIL4 plays important roles in histone modification and piRNA-dependent DNA methylation.

PNLDC1, mouse pre-piRNA Trimmer, is required for meiotic and post-meiotic male germ cell development.

PIWI-interacting RNAs (piRNAs) are germ cell-specific small RNAs essential for retrotransposon gene silencing and male germ cell development. In piRNA biogenesis, the endonuclease MitoPLD/Zucchini cleaves long, single-stranded RNAs to generate 5' termini of precursor piRNAs (pre-piRNAs) that are consecutively loaded into PIWI-family proteins. Subsequently, these pre-piRNAs are trimmed at their 3'-end by an exonuclease called Trimmer. Recently, poly(A)-specific ribonuclease-like domain-containing 1 (PNLDC1) was identified as the pre-piRNA Trimmer in silkworms. However, the function of PNLDC1 in other species remains unknown. Here, we generate Pnldc1 mutant mice and analyze small RNAs in their testes. Our results demonstrate that mouse PNLDC1 functions in the trimming of both embryonic and post-natal pre-piRNAs. In addition, piRNA trimming defects in embryonic and post-natal testes cause impaired DNA methylation and reduced MIWI expression, respectively. Phenotypically, both meiotic and post-meiotic arrests are evident in the same individual Pnldc1 mutant mouse. The former and latter phenotypes are similar to those of MILI and MIWI mutant mice, respectively. Thus, PNLDC1-mediated piRNA trimming is indispensable for the function of piRNAs throughout mouse spermatogenesis.

Mouse GTSF1 is an essential factor for secondary piRNA biogenesis.

The piRNA pathway is a piRNA-guided retrotransposon silencing system which includes processing of retrotransposon transcripts by PIWI-piRNAs in secondary piRNA biogenesis. Although several proteins participate in the piRNA pathway, the ones crucial for the cleavage of target RNAs by PIWI-piRNAs have not been identified. Here, we show that GTSF1, an essential factor for retrotransposon silencing in male germ cells in mice, associates with both MILI and MIWI2, mouse PIWI proteins that function in prospermatogonia. GTSF1 deficiency leads to a severe defect in the production of secondary piRNAs, which are generated from target RNAs of PIWI-piRNAs. Furthermore, in Gtsf1 mutants, a known target RNA of PIWI-piRNAs is left unsliced at the cleavage site, and the generation of secondary piRNAs from this transcript is defective. Our findings indicate that GTSF1 is a crucial factor for the slicing of target RNAs by PIWI-piRNAs and thus affects secondary piRNA biogenesis in prospermatogonia.

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

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.

MIWI2 as an Effector of DNA Methylation and Gene Silencing in Embryonic Male Germ Cells.

During the development of mammalian embryonic germ cells, global demethylation and de novo DNA methylation take place. In mouse embryonic germ cells, two PIWI family proteins, MILI and MIWI2, are essential for the de novo DNA methylation of retrotransposons, presumably through PIWI-interacting RNAs (piRNAs). Although piRNA-associated MIWI2 has been reported to play critical roles in the process, its molecular mechanisms have remained unclear. To identify the mechanism, transgenic mice were produced; they contained a fusion protein of MIWI2 and a zinc finger (ZF) that recognized the promoter region of a type A LINE-1 gene. The ZF-MIWI2 fusion protein brought about DNA methylation, suppression of the type A LINE-1 gene, and a partial rescue of the impaired spermatogenesis of MILI-null mice. In addition, ZF-MIWI2 was associated with the proteins involved in DNA methylation. These data indicate that MIWI2 functions as an effector of de novo DNA methylation of the retrotransposon.

Comprehensive DNA Methylation Analysis of Retrotransposons in Male Germ Cells.

De novo DNA methylation of retrotransposons is critical for silencing. Here, we use DNA methylation analysis to examine retrotransposons in mouse male germ cells. DNA methylation of long interspersed nuclear elements (LINEs) is dependent on piRNA, and younger LINEs exhibit greater piRNA dependence. In contrast, most long terminal repeat (LTR) retrotransposons produce lower levels of piRNAs and do not show significant piRNA dependence. The relationship between DNA methylation and corresponding piRNA expression of several LTR retrotransposons was reduced in Mili-null cells, but not Miwi2-null cells. These observations raise the possibility of piRNA-dependent DNA methylation without Miwi2. Therefore, it appears that the molecular mechanisms of the gene silencing of retrotransposons are more complicated than previously thought.

piRNAs derived from ancient viral processed pseudogenes as transgenerational sequence-specific immune memory in mammals.

Endogenous bornavirus-like nucleoprotein elements (EBLNs) are sequences within vertebrate genomes derived from reverse transcription and integration of ancient bornaviral nucleoprotein mRNA via the host retrotransposon machinery. While species with EBLNs appear relatively resistant to bornaviral disease, the nature of this association is unclear. We hypothesized that EBLNs could give rise to antiviral interfering RNA in the form of PIWI-interacting RNAs (piRNAs), a class of small RNA known to silence transposons but not exogenous viruses. We found that in both rodents and primates, which acquired their EBLNs independently some 25-40 million years ago, EBLNs are present within piRNA-generating regions of the genome far more often than expected by chance alone (ℙ = 8 × 10(-3)-6 × 10(-8)). Three of the seven human EBLNs fall within annotated piRNA clusters and two marmoset EBLNs give rise to bona fide piRNAs. In both rats and mice, at least two of the five EBLNs give rise to abundant piRNAs in the male gonad. While no EBLNs are syntenic between rodent and primate, some of the piRNA clusters containing EBLNs are; thus we deduce that EBLNs were integrated into existing piRNA clusters. All true piRNAs derived from EBLNs are antisense relative to the proposed ancient bornaviral nucleoprotein mRNA. These observations are consistent with a role for EBLN-derived piRNA-like RNAs in interfering with ancient bornaviral infection. They raise the hypothesis that retrotransposon-dependent virus-to-host gene flow could engender RNA-mediated, sequence-specific antiviral immune memory in metazoans analogous to the CRISPR/Cas system in prokaryotes.

Reply to Shoji and Katsuma.

Our paper reports a novel strategy for the artificial introduction of DNA methylation in mouse gonocytes. The manuscript presents data showing that the concomitant expression of sense and anti-sense of EGFP transgenes in embryonic male germ cells induces gene silencing via the piRNA pathway and that the expression of an antisense Dnmt3L transgene induces silencing of the endogenous Dnmt3L gene.

Induction of DNA methylation by artificial piRNA production in male germ cells.

Global DNA demethylation and subsequent de novo DNA methylation take place in mammalian male embryonic germ cells [1-3]. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), which are germline-specific small RNAs, have been postulated to be critically important for de novo DNA methylation of retrotransposon genes, and many proteins, including PIWI family proteins, play pivotal roles in this process [4-6]. In the embryonic mouse testis, two mouse PIWI proteins, mouse PIWI-like (MILI) and mouse PIWI2 (MIWI2), are involved in the biogenesis of piRNAs through the so-called ping-pong amplification cycle [7-10], and long single-stranded RNAs transcribed from the gene regions of piRNA clusters have been proposed to be the initial material [11-16]. However, it remains unclear whether transcription from the piRNA clusters is required for the biogenesis of piRNAs. To answer this question, we developed a novel artificial piRNA production system by simple expression of sense and antisense EGFP mRNAs in embryonic male germ cells in the piRNA biogenesis phase. EGFP expression was silenced by piRNA-dependent DNA methylation, indicating that concomitant expression of sense and antisense RNA transcripts is necessary and sufficient for piRNA production and subsequent piRNA-dependent gene silencing. In addition, we demonstrated that this artificial piRNA induction paradigm could be applied to an endogenous gene essential for spermatogenesis, DNMT3L [3, 17, 18]. This study not only provides novel insights into the molecular mechanisms of piRNA production, but also presents an innovative strategy for inducing epigenetic modification in germ cells.

HSP90α plays an important role in piRNA biogenesis and retrotransposon repression in mouse.

HSP90, found in all kingdoms of life, is a major chaperone protein regulating many client proteins. We demonstrated that HSP90α, one of two paralogs duplicated in vertebrates, plays an important role in the biogenesis of fetal PIWI-interacting RNAs (piRNA), which act against the transposon activities, in mouse male germ cells. The knockout mutation of Hsp90α resulted in a large reduction in the expression of primary and secondary piRNAs and mislocalization of MIWI2, a PIWI homolog. Whereas the mutation in Fkbp6 encoding a co-chaperone reduced piRNAs of 28-32 nucleotides in length, the Hsp90α mutation reduced piRNAs of 24-32 nucleotides, suggesting the presence of both FKBP6-dependent and -independent actions of HSP90α. Although DNA methylation and mRNA levels of L1 retrotransposon were largely unchanged in the Hsp90α mutant testes, the L1-encoded protein was increased, suggesting the presence of post-transcriptional regulation. This study revealed the specialized function of the HSP90α isofom in the piRNA biogenesis and repression of retrotransposons during the development of male germ cells in mammals.

DNA methylation in mouse testes.

DNA methylation of retrotransposons and imprinted genes is accurately regulated in spermatogenesis. In particular, CpG methylation of long interspersed elements-1 (LINE1 or L1) and intracisternal A-particle (IAP) retrotransposons during spermatogenesis has been well characterized. CpG methylation of the regulatory regions of retrotransposons is acquired during embryonic testis development; however, reductions of DNA methylation in LINE1 and/or IAP and/or Rasgrf1, which is an imprinted gene, are observed in deficient mice of piRNA biogenesis concerning. Here, we describe two methods, bisulfite sequencing and Southern blotting using a methylation-sensitive restriction enzyme, for analysis of DNA methylation of LINE1, IAP, and imprinted genes in mouse testes.

The nuage mediates retrotransposon silencing in mouse primordial ovarian follicles.

Mobilization of endogenous retrotransposons can destabilize the genome, an imminent danger during epigenetic reprogramming of cells in the germline. The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway is known to silence retrotransposons in the mouse testes. Several piRNA pathway components localize to the unique, germline structure known as the nuage. In this study, we surveyed mouse ovaries and found, for the first time, transient appearance of nuage-like structures in oocytes of primordial follicles. Mouse vasa homolog (MVH), Piwi-like 2 (PIWIL2/MILI) and tudor domain-containing 9 (TDRD9) are present in these structures, whereas aggregates of germ cell protein with ankyrin repeats, sterile alpha motif and leucine zipper (GASZ) localize separately in the cytoplasm. Retrotransposons are silenced in primordial ovarian follicles, and de-repressed upon reduction of piRNA expression in Mvh, Mili or Gasz mutants. However, these null-mutant females, unlike their male counterparts, are fertile, uncoupling retrotransposon activation from sterility.

GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells.

piRNA (PIWI-interacting RNA) is a germ cell-specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.

Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus.

In germ cells, early embryos, and stem cells of animals, PIWI-interacting RNAs (piRNAs) have an important role in silencing retrotransposons, which are vicious genomic parasites, through transcriptional and post-transcriptional mechanisms. To examine whether the piRNA pathway can be used to silence genes of interest in germ cells, we have generated knock-in mice in which a foreign DNA fragment was inserted into a region generating pachytene piRNAs. The knock-in sequence was transcribed, and the resulting RNA was processed to yield piRNAs in postnatal testes. When reporter genes possessing a sequence complementary to portions of the knock-in sequence were introduced, they were greatly repressed after the time of pachytene piRNA generation. This repression mainly occurred at the post-transcriptional level, as degradation of the reporter RNAs was accelerated. Our results show that the piRNA pathway can be used as a tool for sequence-specific gene silencing in germ cells and support the idea that the piRNA generating regions serve as traps for retrotransposons, enabling the host cell to generate piRNAs against active retrotransposons.

Identification of MIWI-associated Poly(A) RNAs by immunoprecipitation with an anti-MIWI monoclonal antibody.

MIWI is one of the PIWI subfamily of proteins mainly expressed in mouse germ cells, and associates with pachytene piRNAs. MIWI has been thought to play an essential role in spermatogenesis and spermiogenesis via biogenesis and/or stability of pachytene piRNAs, retrotransposon silencing, and post-transcriptional regulation of target mRNAs. However, MIWI's detailed role and function are not well understood. In this study, we produced an anti-MIWI mouse monoclonal antibody and identified MIWI-associated poly(A) RNAs by immunoprecipitation from adult mouse testes lysates. Approximately 70% of the MIWI-associated poly(A) RNAs were known mRNAs and 30% of them were unknown non-coding RNAs. These poly(A) RNAs contained piRNA-encoding RNAs transcribed from piRNA cluster regions and piRNA-encoding mRNA, such as Aym1 mRNA. Mature piRNAs specifically encoded in these piRNA-encoding RNAs were generated in pachytene spermatocytes and not detected in Miwi-deficient (Miwi-/-) testes. Moreover, MIWI associated with a large number of known mRNAs whose expression levels were increased in pachytene spermatocytes, and the expression of these mRNAs was decreased in Miwi-/- testes at 20 days postpartum when pachytene spermatocytes were most abundant. These results strongly suggest that MIWI is involved in pachytene piRNA biogenesis and the positive regulation of target mRNA metabolism in pachytene spermatocytes via association with pachytene piRNA precursors and target mRNAs.

Locus- and domain-dependent control of DNA methylation at mouse B1 retrotransposons during male germ cell development.

In mammals, germ cells undergo striking dynamic changes in DNA methylation during their development. However, the dynamics and mode of methylation are poorly understood for short interspersed elements (SINEs) dispersed throughout the genome. We investigated the DNA methylation status of mouse B1 SINEs in male germ cells at different developmental stages. B1 elements showed a large locus-to-locus variation in methylation; loci close to RNA polymerase II promoters were hypomethylated, while most others were hypermethylated. Interestingly, a mutation that eliminates Piwi-interacting RNAs (piRNAs), which are involved in methylation of long interspersed elements (LINEs), did not affect the level of B1 methylation, implying a piRNA-independent mechanism. Methylation at B1 loci in SINE-poor genomic domains showed a higher dependency on the de novo DNA methyltransferase DNMT3A but not on DNMT3B, suggesting that DNMT3A plays a major role in methylation of these domains. We also found that many genes specifically expressed in the testis possess B1 elements in their promoters, suggesting the involvement of B1 methylation in transcriptional regulation. Taken altogether, our results not only reveal the dynamics and mode of SINE methylation but also suggest how the DNA methylation profile is created in the germline by a pair of DNA methyltransferases.