[Show abstract][Hide abstract] ABSTRACT: 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.
Nucleic Acids Research 09/2014; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fetal oocytes in mammals undergo extensive apoptosis during development. In this issue of Developmental Cell, Malki et al. (2014) provide insight into how and why such massive oocyte loss occurs through the demonstration that the expression level of LINE-1 retrotransposon defines the survival threshold and thus viability of fetal oocytes.
[Show abstract][Hide abstract] ABSTRACT: Piwi-interacting RNAs (piRNAs) are gonad-specific small RNAs that provide defense against transposable genetic elements called transposons. Our knowledge of piRNA biogenesis is sketchy, partly due to an incomplete inventory of the factors involved. Here, we identify Tudor domain-containing 12 (TDRD12; also known as ECAT8) as a unique piRNA biogenesis factor in mice. TDRD12 is detected in complexes containing Piwi protein MILI (PIWIL2), its associated primary piRNAs, and TDRD1, all of which are already implicated in secondary piRNA biogenesis. Male mice carrying either a nonsense point mutation (reproductive mutant 23 or repro23 mice) or a targeted deletion in the Tdrd12 locus are infertile and derepress retrotransposons. We find that TDRD12 is dispensable for primary piRNA biogenesis but essential for production of secondary piRNAs that enter Piwi protein MIWI2 (PIWIL4). Cell-culture studies with the insect ortholog of TDRD12 suggest a role for the multidomain protein in mediating complex formation with other participants during secondary piRNA biogenesis.
Proceedings of the National Academy of Sciences 09/2013; 110(41). · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Transposable elements and their fossil sequences occupy about half of the genome in mammals. While most of these selfish mobile elements have been inactivated by truncations and mutations during evolution, some copies remain competent to transpose and/or amplify, posing an ongoing genetic threat. To control such mutagenic sequences, host genomes have developed multiple layers of defence mechanisms, including epigenetic regulation and RNA silencing. Germ cells, in particular, employ the piwi-small RNA pathway, which plays a central and adaptive role in safeguarding the germline genome from retrotransposons. Recent studies have revealed that a class of developmentally regulated genes, which have long been implicated in germ cell specification and differentiation, such as vasa and tudor family genes, play key roles in the piwi pathway to suppress retrotransposons, indicating that the piwi-mediated genome protection is at the core of germline development. Furthermore, while the piwi system primarily operates post-transcriptionally at the RNA level, it also affects the epigenetics of cognate genome loci, offering an intriguing link between small RNAs and transcriptional control in mammals. In this review, we summarize our current understanding of the piwi pathway in mice, which is emerging as a fundamental component of spermatogenesis that ensures male fertility and genome integrity.
Philosophical Transactions of The Royal Society B Biological Sciences 01/2013; 368(1609):20110338. · 6.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epigenetic silencing of transposons by Piwi-interacting RNAs (piRNAs) constitutes an RNA-based genome defense mechanism. Piwi endonuclease action amplifies the piRNA pool by generating new piRNAs from target transcripts by a poorly understood mechanism. Here, we identified mouse Fkbp6 as a factor in this biogenesis pathway delivering piRNAs to the Piwi protein Miwi2. Mice lacking Fkbp6 derepress LINE1 (L1) retrotransposon and display reduced DNA methylation due to deficient nuclear accumulation of Miwi2. Like other cochaperones, Fkbp6 associates with the molecular chaperone Hsp90 via its tetratricopeptide repeat (TPR) domain. Inhibition of the ATP-dependent Hsp90 activity in an insect cell culture model results in the accumulation of short antisense RNAs in Piwi complexes. We identify these to be byproducts of piRNA amplification that accumulate only in nuage-localized Piwi proteins. We propose that the chaperone machinery normally ejects these inhibitory RNAs, allowing turnover of Piwi complexes for their continued participation in piRNA amplification.
[Show abstract][Hide abstract] ABSTRACT: Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs expressed in the animal gonads. They are implicated in silencing the genome instability threat posed by mobile genetic elements called transposons. Unlike other small RNAs, which use double-stranded precursors, piRNAs seem to arise from long single-stranded precursor transcripts expressed from discrete genomic regions. In mice, the Piwi pathway is essential for male fertility, and its loss-of-function mutations affect several distinct stages of spermatogenesis. While this small RNA pathway primarily operates post-transcriptionally, it also impacts DNA methylation of target retrotransposon loci, representing an intriguing model of RNA-directed epigenetic control in mammals. Remarkably the Piwi pathway components are specifically localized at germinal granule/nuage, an evolutionarily conserved but still enigmatic ribonucleoprotein compartment in the germline. The inaccessibility of the germline for easy experimental manipulation has meant that this class of RNAs has remained enigmatic. However, recent advances in the use of cell culture models and cell-free systems have greatly advanced our understanding. In this review, we briefly summarize our current understanding of the Piwi pathway, focusing on its developmental regulation, piRNA biogenesis and key function in male germline development from fetal spermatogonial stem cell stage to postnatal haploid spermiogenesis in mice.
[Show abstract][Hide abstract] ABSTRACT: PSF is considered to have multiple functions in RNA processing, transcription and DNA repair by mitotic recombination. In the present study, we found that PSF is produced in spermatogonia, spermatocytes and spermatids, suggesting that PSF may also function in meiotic recombination. We tested the effect of PSF on homologous pairing by the meiosis-specific recombinase DMC1, and found that human PSF robustly stimulated it. PSF synergistically enhanced the formation of a synaptic complex containing DMC1, ssDNA and dsDNA during homologous pairing. The PSF-mediated DMC1 stimulation may be promoted by its DNA aggregation activity, which increases the local concentrations of ssDNA and dsDNA for homologous pairing by DMC1. These results suggested that PSF may function as an activator for the meiosis-specific recombinase DMC1 in higher eukaryotes.
Nucleic Acids Research 12/2011; 40(7):3031-41. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Repetitive-element-derived Piwi-interacting RNAs (piRNAs) act together with Piwi proteins Mili (also known as Piwil2) and Miwi2 (also known as Piwil4) in a genome defence mechanism that initiates transposon silencing via DNA methylation in the mouse male embryonic germ line. This silencing depends on the participation of the Piwi proteins in a slicer-dependent piRNA amplification pathway and is essential for male fertility. A third Piwi family member, Miwi (also known as Piwil1), is expressed in specific postnatal germ cells and associates with a unique set of piRNAs of unknown function. Here we show that Miwi is a small RNA-guided RNase (slicer) that requires extensive complementarity for target cleavage in vitro. Disruption of its catalytic activity in mice by a single point mutation causes male infertility, and mutant germ cells show increased accumulation of LINE1 retrotransposon transcripts. We provide evidence for Miwi slicer activity directly cleaving transposon messenger RNAs, offering an explanation for the continued maintenance of repeat-derived piRNAs long after transposon silencing is established in germline stem cells. Furthermore, our study supports a slicer-dependent silencing mechanism that functions without piRNA amplification. Thus, Piwi proteins seem to act in a two-pronged mammalian transposon silencing strategy: one promotes transcriptional repression in the embryo, the other reinforces silencing at the post-transcriptional level after birth.
[Show abstract][Hide abstract] ABSTRACT: In the male germline in mammals, chromatoid bodies, a specialized assembly of cytoplasmic ribonucleoprotein (RNP), are structurally evident during meiosis and haploidgenesis, but their developmental origin and regulation remain elusive. The tudor domain containing proteins constitute a conserved class of chromatoid body components. We show that tudor domain containing 7 (Tdrd7), the deficiency of which causes male sterility and age-related cataract (as well as glaucoma), is essential for haploid spermatid development and defines, in concert with Tdrd6, key biogenesis processes of chromatoid bodies. Single and double knockouts of Tdrd7 and Tdrd6 demonstrated that these spermiogenic tudor genes orchestrate developmental programs for ordered remodeling of chromatoid bodies, including the initial establishment, subsequent RNP fusion with ubiquitous processing bodies/GW bodies and later structural maintenance. Tdrd7 suppresses LINE1 retrotransposons independently of piwi-interacting RNA (piRNA) biogenesis wherein Tdrd1 and Tdrd9 operate, indicating that distinct Tdrd pathways act against retrotransposons in the male germline. Tdrd6, in contrast, does not affect retrotransposons but functions at a later stage of spermiogenesis when chromatoid bodies exhibit aggresome-like properties. Our results delineate that chromatoid bodies assemble as an integrated compartment incorporating both germline and ubiquitous features as spermatogenesis proceeds and that the conserved tudor family genes act as master regulators of this unique RNP remodeling, which is genetically linked to the male germline integrity in mammals.
Proceedings of the National Academy of Sciences 06/2011; 108(26):10579-84. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MITOPLD is a member of the phospholipase D superfamily proteins conserved among diverse species. Zucchini (Zuc), the Drosophila homolog of MITOPLD, has been implicated in primary biogenesis of Piwi-interacting RNAs (piRNAs). By contrast, MITOPLD has been shown to hydrolyze cardiolipin in the outer membrane of mitochondria to generate phosphatidic acid, which is a signaling molecule. To assess whether the mammalian MITOPLD is involved in piRNA biogenesis, we generated Mitopld mutant mice. The mice display meiotic arrest during spermatogenesis, demethylation and derepression of retrotransposons, and defects in primary piRNA biogenesis. Furthermore, in mutant germ cells, mitochondria and the components of the nuage, a perinuclear structure involved in piRNA biogenesis/function, are mislocalized to regions around the centrosome, suggesting that MITOPLD may be involved in microtubule-dependent localization of mitochondria and these proteins. Our results indicate a conserved role for MITOPLD/Zuc in the piRNA pathway and link mitochondrial membrane metabolism/signaling to small RNA biogenesis.
[Show abstract][Hide abstract] ABSTRACT: The Tudor domain-containing proteins (TDRDs) are an evolutionarily conserved family of proteins involved in germ cell development. We show here that in mice, TDRD5 is a novel component of the intermitochondrial cements (IMCs) and the chromatoid bodies (CBs), which are cytoplasmic ribonucleoprotein granules involved in RNA processing for spermatogenesis. Tdrd5-deficient males are sterile because of spermiogenic arrest at the round spermatid stage, with occasional failure in meiotic prophase. Without TDRD5, IMCs and CBs are disorganized, with mislocalization of their key components, including TDRD1/6/7/9 and MIWI/MILI/MIWI2. In addition, Tdrd5-deficient germ cells fail to repress LINE-1 retrotransposons with DNA-demethylated promoters. Cyclic adenosine monophosphate response element modulator (CREM) and TRF2, key transcription factors for spermiogenesis, are expressed in Tdrd5-deficient round spermatids, but their targets, including Prm1/Prm2/Tnp1, are severely down-regulated, which indicates the importance of IMC/CB-mediated regulation for postmeiotic gene expression. Strikingly, Tdrd5-deficient round spermatids injected into oocytes contribute to fertile offspring, demonstrating that acquisition of a functional haploid genome may be uncoupled from TDRD5 function.
The Journal of Cell Biology 03/2011; 192(5):781-95. · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: VASA is an evolutionarily conserved RNA helicase essential for germ cell development. The mouse PIWI family proteins MILI and MIWI2 are involved in production of Piwi-interacting RNAs (piRNAs) in fetal male germ cells through a ping-pong amplification cycle. Expression of retrotransposons is elevated in MILI- and MIWI2-deficient male germ cells due to defective de novo DNA methylation, which is presumably caused by impaired piRNA expression. Here, we report that essentially the same abnormalities are observed in MVH (mouse VASA homolog)-deficient mice. Comprehensive analysis of piRNAs in MVH-deficient fetal male germ cells showed that MVH plays crucial roles in the early phase of the ping-pong amplification cycle.
Genes & development 05/2010; 24(9):887-92. · 12.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Host-defense mechanisms against transposable elements are critical to protect the genome information. Here we show that tudor-domain containing 9 (Tdrd9) is essential for silencing Line-1 retrotransposon in the mouse male germline. Tdrd9 encodes an ATPase/DExH-type helicase, and its mutation causes male sterility showing meiotic failure. In Tdrd9 mutants, Line-1 was highly activated and piwi-interacting small RNAs (piRNAs) corresponding to Line-1 were increased, suggesting that feedforward amplification operates in the mutant. In fetal testes, Tdrd9 mutation causes Line-1 desilencing and an aberrant piRNA profile in prospermatogonia, followed by cognate DNA demethylation. TDRD9 complexes with MIWI2 with distinct compartmentalization in processing bodies, and this TDRD9-MIWI2 localization is regulated by MILI and TDRD1 residing at intermitochondrial cement. Our results identify TDRD9 as a functional partner of MIWI2 and indicate that the tudor-piwi association is a conserved feature, while two separate axes, TDRD9-MIWI2 and TDRD1-MILI, cooperate nonredundantly in the piwi-small RNA pathway in the mouse male germline.
[Show abstract][Hide abstract] ABSTRACT: Spermatogonial stem cells (SSCs) continuously undergo self-renewal division to support spermatogenesis. SSCs are thought to have a fixed phenotype, and development of a germ cell transplantation technique facilitated their characterization and prospective isolation in a deterministic manner; however, our in vitro SSC culture experiments indicated heterogeneity of cultured cells and suggested that they might not follow deterministic fate commitment in vitro.
In this study, we report phenotypic plasticity of SSCs. Although c-kit tyrosine kinase receptor (Kit) is not expressed in SSCs in vivo, it was upregulated when SSCs were cultured on laminin in vitro. Both Kit(-) and Kit(+) cells in culture showed comparable levels of SSC activity after germ cell transplantation. Unlike differentiating spermatogonia that depend on Kit for survival and proliferation, Kit expressed on SSCs did not play any role in SSC self-renewal. Moreover, Kit expression on SSCs changed dynamically once proliferation began after germ cell transplantation in vivo.
These results indicate that SSCs can change their phenotype according to their microenvironment and stochastically express Kit. Our results also suggest that activated and non-activated SSCs show distinct phenotypes.
PLoS ONE 11/2009; 4(11):e7909. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mouse Piwi family proteins (MILI, MIWI and MIWI2) play pivotal roles in spermatogenesis through transcriptional and post-transcriptional gene regulation. To reveal the molecular functions of these proteins, we investigate the proteins that bind to MILI in adult mouse testes. We found that both MILI and MIWI bind to TDRD1/MTR-1, which is also an essential protein for spermatogenesis. Co-immunoprecipitation assays and subcellular localization of the proteins and mutants thereof revealed a complex formation involving MILI, MIWI and TDRD1/MTR-1. In addition, the subcellular localizations of MILI and TDRD1/MTR-1 were altered, and chromatoid body formation was impaired in the MIWI-null round spermatids. These data suggest that the formation of complexes between MILI, MIWI and TDRD1/MTR-1 is critical for the integrated subcellular localizations of these proteins, and is presumably essential for spermatogenesis.
Genes to Cells 10/2009; 14(10):1155-65. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations of RNA-binding proteins such as NANOS3, TIAL1, and DND1 in mice have been known to result in the failure of survival and/or proliferation of primordial germ cells (PGCs) soon after their fate is specified (around embryonic day (E) 8.0), leading to the infertility of these animals. However, the mechanisms of actions of these RNA-binding proteins remain largely unresolved. As a foundation to explore the role of these RNA-binding proteins in germ cells, we established a novel transgenic reporter strain that expresses NANOS3 fused with EGFP under the control of Nanos3 regulatory elements. NANOS3-EGFP exhibited exclusive expression in PGCs as early as E7.25, and continued to be expressed in female germ cells until around E14.5 and in male germ cells throughout the fetal period with declining expression levels after E16.5. NANOS3-EGFP resumed strong expression in postnatal spermatogonia and continued to be expressed in undifferentiated spermatogonial cells in adults. Importantly, the Nanos3-EGFP transgene rescued the sterile phenotype of Nanos3 homozygous mutants, demonstrating the functional equivalency of NANOS3-EGFP with endogenous NANOS3. We found that throughout germ cell development, a predominant amount of NANOS3-EGFP co-localized with TIAL1 (also known as TIAR) and phosphorylated eukaryotic initiation factor 2alpha, markers for the stress granules, whereas a fraction of it showed co-localization with DCP1A, a marker for the processing bodies. On the other hand, NANOS3-EGFP did not co-localize with Tudor domain-containing protein 1, a marker for the intermitochondrial cements, in spermatogenic cells. These findings unveil the presence of distinct posttranscriptional regulations in PGCs soon after their specification, for which RNA-binding proteins such as NANOS3 and TIAL1 would play critical functions.
[Show abstract][Hide abstract] ABSTRACT: In germ cells, Piwi proteins interact with a specific class of small noncoding RNAs, piwi-interacting RNAs (piRNAs). Together, these form a pathway that represses transposable elements, thus safeguarding germ cell genomes. Basic models describe the overall operation of piRNA pathways. However, the protein compositions of Piwi complexes, the critical protein-protein interactions that drive small RNA production and target recognition, and the precise molecular consequences of conserved localization to germline structures, call nuage, remains poorly understood. We purified the three murine Piwi family proteins, MILI, MIWI, and MIWI2, from mouse germ cells and characterized their interacting protein partners. Piwi proteins were found in complex with PRMT5/WDR77, an enzyme that dimethylates arginine residues. By immunoprecipitation with specific antibodies and by mass spectrometry, we found that Piwi proteins are arginine methylated at conserved positions in their N termini. These modifications are essential to direct complex formation with specific members of the Tudor protein family. Recognition of methylarginine marks by Tudor proteins can drive the localization of Piwi proteins to cytoplasmic foci in an artificial setting, supporting a role for this interaction in Piwi localization to nuage, a characteristic that correlates with proper operation of the piRNA pathway and transposon silencing in multiple organisms.
Genes & development 09/2009; 23(15):1749-62. · 12.64 Impact Factor