RESUMEN
The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.
Asunto(s)
Cromatina/metabolismo , Protaminas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/fisiología , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Fertilización/genética , Histonas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/metabolismo , Oocitos/fisiología , Fosforilación , Protamina Quinasa/genética , Protamina Quinasa/metabolismo , Protaminas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Empalme del ARN/genética , Empalme del ARN/fisiología , Espermatozoides/metabolismo , Factores de Transcripción/metabolismo , Cigoto/metabolismoRESUMEN
Spermiogenesis is a highly orchestrated developmental process during which chromatin condensation decouples transcription from translation. Spermiogenic mRNAs are transcribed earlier and stored in a translationally inert state until needed for translation; however, it remains largely unclear how such repressed mRNAs become activated during spermiogenesis. We previously reported that the MIWI/piRNA machinery is responsible for mRNA elimination during late spermiogenesis in preparation for spermatozoa production. Here we unexpectedly discover that the same machinery is also responsible for activating translation of a subset of spermiogenic mRNAs to coordinate with morphological transformation into spermatozoa. Such action requires specific base-pairing interactions of piRNAs with target mRNAs in their 3' UTRs, which activates translation through coupling with cis-acting AU-rich elements to nucleate the formation of a MIWI/piRNA/eIF3f/HuR super-complex in a developmental stage-specific manner. These findings reveal a critical role of the piRNA system in translation activation, which we show is functionally required for spermatid development.
Asunto(s)
Proteínas Argonautas/metabolismo , Iniciación de la Cadena Peptídica Traduccional , ARN Interferente Pequeño/metabolismo , Espermatogénesis , Regiones no Traducidas 3' , Animales , Proteínas Argonautas/genética , Emparejamiento Base , Células Cultivadas , Proteína 1 Similar a ELAV/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Genetic studies have elucidated critical roles of Piwi proteins in germline development in animals, but whether Piwi is an actual disease gene in human infertility remains unknown. We report germline mutations in human Piwi (Hiwi) in patients with azoospermia that prevent its ubiquitination and degradation. By modeling such mutations in Piwi (Miwi) knockin mice, we demonstrate that the genetic defects are directly responsible for male infertility. Mechanistically, we show that MIWI binds the histone ubiquitin ligase RNF8 in a Piwi-interacting RNA (piRNA)-independent manner, and MIWI stabilization sequesters RNF8 in the cytoplasm of late spermatids. The resulting aberrant sperm show histone retention, abnormal morphology, and severely compromised activity, which can be functionally rescued via blocking RNF8-MIWI interaction in spermatids with an RNF8-N peptide. Collectively, our findings identify Piwi as a factor in human infertility and reveal its role in regulating the histone-to-protamine exchange during spermiogenesis.
Asunto(s)
Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Azoospermia/genética , Mutación , Animales , Azoospermia/metabolismo , Cromatina/metabolismo , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Histonas/metabolismo , Humanos , Intrones , Masculino , Ratones , Linaje , Protaminas/metabolismo , Proteolisis , Espermatogénesis , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
PIWI proteins and PIWI-interacting RNAs (piRNAs) are specifically expressed in animal germlines and play essential roles during gametogenesis in animals. The primary function of PIWI/piRNAs is known to silence transposable elements for protecting genome integrity in animal germlines, while their roles beyond silencing transposons are also documented by us and others. In particular, we show that mouse PIWIL1 (MIWI)/piRNAs play a dual role in regulating protein-coding genes in mouse spermatids through interacting with different protein factors in a developmental stage-dependent manner, including translationally activating a subset of AU-rich element-containing mRNAs in round spermatids and inducing massive mRNA degradation in late spermatids. We further show that MIWI is eliminated through the ubiquitin-26S proteasome pathway during late spermiogenesis. By exploring the biological function of MIWI ubiquitination by APC/C, we identified ubiquitination-deficient mutations in human PIWIL1 of infertile men and further established their causative role in male infertility in mouse model, supporting PIWIL1 as a human male infertility-relevant gene. Additionally, we reported that PIWIL1, aberrantly induced in human tumors, functions as an oncoprotein in a piRNA-independent manner in cancer cells. In the current review, we summarize our latest findings regarding the roles and mechanisms of PIWIL1 and piRNAs in mouse spermatids and human diseases, and discuss the related works in the field.
Asunto(s)
Proteínas Argonautas , Infertilidad Masculina , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Células Germinativas/metabolismo , Humanos , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Masculino , Ratones , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Espermátides/metabolismo , Espermatogénesis/genéticaRESUMEN
Heart performance relies on highly coordinated excitation-contraction (EC) coupling, and defects in this critical process may be exacerbated by additional genetic defects and/or environmental insults to cause eventual heart failure. Here we report a regulatory pathway consisting of the RNA binding protein RBFox2, a stress-induced microRNA miR-34a, and the essential EC coupler JPH2. In this pathway, initial cardiac defects diminish RBFox2 expression, which induces transcriptional repression of miR-34a, and elevated miR-34a targets Jph2 to impair EC coupling, which further manifests heart dysfunction, leading to progressive heart failure. The key contribution of miR-34a to this process is further established by administrating its mimic, which is sufficient to induce cardiac defects, and by using its antagomir to alleviate RBFox2 depletion-induced heart dysfunction. These findings elucidate a potential feed-forward mechanism to account for a critical transition to cardiac decompensation and suggest a potential therapeutic avenue against heart failure.
Asunto(s)
Insuficiencia Cardíaca/metabolismo , Corazón/fisiopatología , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Proteínas Musculares/metabolismo , Factores de Empalme de ARN/metabolismo , Animales , Regulación hacia Abajo , Insuficiencia Cardíaca/fisiopatología , Humanos , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiologíaRESUMEN
Small non-coding RNAs (sncRNAs), such as microRNAs (miRNAs), small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), and transfer RNA (tRNA)-derived small RNAs (tsRNAs), play essential roles in regulating various cellular and developmental processes. Over the past three decades, researchers have identified novel sncRNA species from various organisms. These molecules demonstrate dynamic expression and diverse functions, and they are subject to intricate regulation through RNA modifications in both healthy and diseased states. Notably, certain sncRNAs in gametes, particularly sperm, respond to environmental stimuli and facilitate epigenetic inheritance. Collectively, the in-depth understanding of sncRNA functions and mechanisms has accelerated the development of small RNA-based therapeutics. In this review, we present the recent advances in the field, including new sncRNA species and the regulatory influences of RNA modifications. We also discuss the current limitations and challenges associated with using small RNAs as either biomarkers or therapeutic drugs.
RESUMEN
PIWI-clade proteins harness piRNAs of 24-33 nt in length. Of great puzzles are how PIWI-clade proteins incorporate piRNAs of different sizes and whether the size matters to PIWI/piRNA function. Here we report that a PIWI-Ins module unique in PIWI-clade proteins helps define the length of piRNAs. Deletion of PIWI-Ins in Miwi shifts MIWI to load with shorter piRNAs and causes spermiogenic failure in mice, demonstrating the functional importance of this regulatory module. Mechanistically, we show that longer piRNAs provide additional complementarity to target mRNAs, thereby enhancing the assembly of the MIWI/eIF3f/HuR super-complex for translational activation. Importantly, we identify a c.1108C>T (p.R370W) mutation of HIWI (human PIWIL1) in infertile men and demonstrate in Miwi knock-in mice that this genetic mutation impairs male fertility by altering the property of PIWI-Ins in selecting longer piRNAs. These findings reveal a critical role of PIWI-Ins-ensured longer piRNAs in fine-tuning MIWI/piRNA targeting capacity, proven essential for spermatid development and male fertility.
Asunto(s)
ARN de Interacción con Piwi , Testículo , Humanos , Masculino , Ratones , Animales , Testículo/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Espermatogénesis/genética , Proteínas/metabolismo , Fertilidad/genética , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismoRESUMEN
Postmeiotic spermatids use a unique strategy to coordinate gene expression with morphological transformation, in which transcription and translation take place at separate developmental stages, but how mRNAs stored as translationally inert messenger ribonucleoproteins in developing spermatids become activated remains largely unknown. Here, we report that the RNA binding protein FXR1, a member of the fragile X-related (FXR) family, is highly expressed in late spermatids and undergoes liquid-liquid phase separation (LLPS) to merge messenger ribonucleoprotein granules with the translation machinery to convert stored mRNAs into a translationally activated state. Germline-specific Fxr1 ablation in mice impaired the translation of target mRNAs and caused defective spermatid development and male infertility, and a phase separation-deficient FXR1L351P mutation in Fxr1 knock-in mice produced the same developmental defect. These findings uncover a mechanism for translational reprogramming with LLPS as a key driver in spermiogenesis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Biosíntesis de Proteínas , ARN Mensajero Almacenado , Proteínas de Unión al ARN , Espermátides , Espermatogénesis , Animales , Infertilidad Masculina/genética , Masculino , Ratones , ARN Mensajero Almacenado/genética , ARN Mensajero Almacenado/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Espermátides/crecimiento & desarrollo , Espermátides/metabolismo , Espermatogénesis/genéticaRESUMEN
Infertility has been reported as one of the most common reproductive impairments, affecting nearly one in six couples worldwide. A large proportion of infertility cases are diagnosed as idiopathic, signifying a deficit in information surrounding the pathology of infertility and necessity of medical intervention such as assisted reproductive therapy. Small noncoding RNAs (sncRNAs) are well-established regulators of mammalian reproduction. Advanced technologies have revealed the dynamic expression and diverse functions of sncRNAs during mammalian germ cell development. Mounting evidence indicates sncRNAs in sperm, especially microRNAs (miRNAs) and transfer RNA (tRNA)-derived small RNAs (tsRNAs), are sensitive to environmental changes and mediate the inheritance of paternally acquired metabolic and mental traits. Here, we review the critical roles of sncRNAs in mammalian germ cell development. Furthermore, we highlight the functions of sperm-borne sncRNAs in epigenetic inheritance. We also discuss evidence supporting sncRNAs as promising biomarkers for fertility and embryo quality in addition to the present limitations of using sncRNAs for infertility diagnosis and treatment.
RESUMEN
The mammalian epididymis not only plays a fundamental role in the maturation of spermatozoa, but also provides protection against various stressors. The foremost among these is the threat posed by oxidative stress, which arises from an imbalance in reactive oxygen species and can elicit damage to cellular lipids, proteins, and nucleic acids. In mice, the risk of oxidative damage to spermatozoa is mitigated through the expression and secretion of glutathione peroxidase 5 (GPX5) as a major luminal scavenger in the proximal caput epididymidal segment. Accordingly, the loss of GPX5-mediated protection leads to impaired DNA integrity in the spermatozoa of aged Gpx5-/- mice. To explore the underlying mechanism, we have conducted transcriptomic analysis of caput epididymidal epithelial cells from aged (13 months old) Gpx5-/- mice. This analysis revealed the dysregulation of several thousand epididymal mRNA transcripts, including the downregulation of a subgroup of piRNA pathway genes, in aged Gpx5-/- mice. In agreement with these findings, we also observed the loss of piRNAs, which potentially bind to the P-element-induced wimpy testis (PIWI)-like proteins PIWIL1 and PIWIL2. The absence of these piRNAs was correlated with the elevated mRNA levels of their putative gene targets in the caput epididymidis of Gpx5-/- mice. Importantly, the oxidative stress response genes tend to have more targeting piRNAs, and many of them were among the top increased genes upon the loss of GPX5. Taken together, our findings suggest the existence of a previously uncharacterized somatic piRNA pathway in the mammalian epididymis and its possible involvement in the aging and oxidative stress-mediated responses.
Asunto(s)
Epidídimo/metabolismo , Glutatión Peroxidasa/fisiología , ARN Interferente Pequeño/metabolismo , Envejecimiento/metabolismo , Animales , Regulación hacia Abajo , Epidídimo/enzimología , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Glutatión Peroxidasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Higher eukaryotic genomes are bound by a large number of coding and non-coding RNAs, but approaches to comprehensively map the identity and binding sites of these RNAs are lacking. Here we report a method to capture in situ global RNA interactions with DNA by deep sequencing (GRID-seq), which enables the comprehensive identification of the entire repertoire of chromatin-interacting RNAs and their respective binding sites. In human, mouse, and Drosophila cells, we detected a large set of tissue-specific coding and non-coding RNAs that are bound to active promoters and enhancers, especially super-enhancers. Assuming that most mRNA-chromatin interactions indicate the physical proximity of a promoter and an enhancer, we constructed a three-dimensional global connectivity map of promoters and enhancers, revealing transcription-activity-linked genomic interactions in the nucleus.
Asunto(s)
Cromatina/metabolismo , ADN/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/metabolismo , Animales , Línea Celular Tumoral , Drosophila , Elementos de Facilitación Genéticos , Humanos , Regiones Promotoras Genéticas , Reproducibilidad de los ResultadosAsunto(s)
Proteínas Argonautas , ARN Interferente Pequeño , Espermatogénesis , Animales , Masculino , Ratones , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Células Germinativas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Espermatogénesis/genética , Testículo/metabolismoRESUMEN
MicroRNA (miRNA) biogenesis is known to be modulated by a variety of RNA-binding proteins (RBPs), but in most cases, individual RBPs appear to influence the processing of a small subset of target miRNAs. Here, we report that the RNA-binding NONO-PSF heterodimer binds a large number of expressed pri-miRNAs in HeLa cells to globally enhance pri-miRNA processing by the Drosha-DGCR8 Microprocessor. NONO and PSF are key components of paraspeckles organized by the long noncoding RNA (lncRNA) NEAT1. We further demonstrate that NEAT1 also has a profound effect on global pri-miRNA processing. Mechanistic dissection reveals that NEAT1 broadly interacts with the NONO-PSF heterodimer as well as many other RBPs and that multiple RNA segments in NEAT1, including a 'pseudo pri-miRNA' near its 3' end, help attract the Microprocessor. These findings suggest a 'bird nest' model in which an lncRNA orchestrates efficient processing of potentially an entire class of small noncoding RNAs in the nucleus.
Asunto(s)
MicroARNs/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Factores de Transcripción de Octámeros/metabolismo , Factor de Empalme Asociado a PTB/metabolismo , Procesamiento Postranscripcional del ARN , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismo , Proteínas de Unión al ADN , Células HeLa , Humanos , Unión ProteicaRESUMEN
BACKGROUND: Young neurons in the developing brain establish a polarized morphology for proper migration. The PIWI family of piRNA processing proteins are considered to be restrictively expressed in germline tissues and several types of cancer cells. They play important roles in spermatogenesis, stem cell maintenance, piRNA biogenesis, and transposon silencing. Interestingly a recent study showed that de novo mutations of PIWI family members are strongly associated with autism. RESULTS: Here, we report that PIWI-like 1 (PIWIL1), a PIWI family member known to be essential for the transition of round spermatid into elongated spermatid, plays a role in the polarization and radial migration of newborn neurons in the developing cerebral cortex. Knocking down PIWIL1 in newborn cortical neurons by in utero electroporation of specific siRNAs resulted in retardation of the transition of neurons from the multipolar stage to the bipolar stage followed by a defect in their radial migration to the proper destination. Domain analysis showed that both the RNA binding PAZ domain and the RNA processing PIWI domain in PIWIL1 were indispensable for its function in neuronal migration. Furthermore, we found that PIWIL1 unexpectedly regulates the expression of microtubule-associated proteins in cortical neurons. CONCLUSIONS: PIWIL1 regulates neuronal polarization and radial migration partly via modulating the expression of microtubule-associated proteins (MAPs). Our finding of PIWIL1's function in neuronal development implies conserved functions of molecules participating in morphogenesis of brain and germline tissue and provides a mechanism as to how mutations of PIWI may be associated with autism.
Asunto(s)
Proteínas Argonautas/metabolismo , Movimiento Celular , Polaridad Celular , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/citología , Animales , Corteza Cerebral/citología , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Metilación de ADN/genética , Técnicas de Silenciamiento del Gen , Humanos , Ratones Endogámicos C57BL , Mitosis , Neuronas/metabolismo , Estructura Terciaria de Proteína , Estabilidad del ARN , Ratas Sprague-DawleyRESUMEN
The piRNA machinery is known for its role in mediating epigenetic silencing of transposons. Recent studies suggest that this function also involves piRNA-guided cleavage of transposon-derived transcripts. As many piRNAs also appear to have the capacity to target diverse mRNAs, this raises the intriguing possibility that piRNAs may act extensively as siRNAs to degrade specific mRNAs. To directly test this hypothesis, we compared mouse PIWI (MIWI)-associated piRNAs with experimentally identified cleaved mRNA fragments from mouse testes, and observed cleavage sites that predominantly occur at position 10 from the 5' end of putative targeting piRNAs. We also noted strong biases for U and A residues at nucleotide positions 1 and 10, respectively, in both piRNAs and mRNA fragments, features that resemble the pattern of piRNA amplification by the 'ping-pong' cycle. Through mapping of MIWI-RNA interactions by CLIP-seq and gene expression profiling, we found that many potential piRNA-targeted mRNAs directly interact with MIWI and show elevated expression levels in the testes of Miwi catalytic mutant mice. Reporter-based assays further revealed the importance of base pairing between piRNAs and mRNA targets and the requirement for both the slicer activity and piRNA-loading ability of MIWI in piRNA-mediated target repression. Importantly, we demonstrated that proper turnover of certain key piRNA targets is essential for sperm formation. Together, these findings reveal the siRNA-like function of the piRNA machinery in mouse testes and its central requirement for male germ cell development and maturation.
Asunto(s)
Proteínas Argonautas/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Testículo/metabolismo , Regiones no Traducidas 3' , Animales , Proteínas Argonautas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Exonucleasas/genética , Exonucleasas/metabolismo , Masculino , Ratones , División del ARN , ARN Interferente Pequeño/genética , Análisis de Secuencia de ARN , Espermatogénesis , Espermatozoides/citologíaRESUMEN
Small noncoding RNAs (ncRNAs) are a novel class of gene regulators that modulate gene expression at transcriptional, post-transcriptional, and epigenetic levels, and they play crucial roles in almost all cellular processes in eukaryotes. Recent studies have indicated that several types of small noncoding RNAs, including microRNAs (miRNAs), endo-small interference RNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs), are expressed in the male germline and are required for spermatogenesis in animals. In this review, we summarize the recent knowledge of these small noncoding RNAs in male germ cells and their biological functions and mechanisms of action in animal spermatogenesis.
Asunto(s)
Regulación de la Expresión Génica , Infertilidad Masculina , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Espermatogénesis , Animales , Humanos , MasculinoRESUMEN
Spermatogenesis in mammals is characterized by two waves of piRNA expression: one corresponds to classic piRNAs responsible for silencing retrotransponsons and the second wave is predominantly derived from nontransposon intergenic regions in pachytene spermatocytes, but the function of these pachytene piRNAs is largely unknown. Here, we report the involvement of pachytene piRNAs in instructing massive mRNA elimination in mouse elongating spermatids (ES). We demonstrate that a piRNA-induced silencing complex (pi-RISC) containing murine PIWI (MIWI) and deadenylase CAF1 is selectively assembled in ES, which is responsible for inducing mRNA deadenylation and decay via a mechanism that resembles the action of miRNAs in somatic cells. Such a highly orchestrated program appears to take full advantage of the enormous repertoire of diversified targeting capacity of pachytene piRNAs derived from nontransposon intergenic regions. These findings suggest that pachytene piRNAs are responsible for inactivating vast cellular programs in preparation for sperm production from ES.
Asunto(s)
ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Espermatogénesis , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Exorribonucleasas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Proteínas/metabolismo , Proteínas Represoras , Ribonucleasas , Alineación de Secuencia , Espermátides/citología , Espermátides/metabolismoRESUMEN
The PIWI/PIWI-interacting RNA (piRNA) machinery has been well documented to maintain genome integrity by silencing transposons in animal germ cells. Recent studies have advanced our understanding of the biogenesis and function of this machinery; however, its metabolism has remained largely unexplored. Here, we show that murine PIWI (MIWI) is degraded through the APC/C-26S proteasome pathway and that piRNAs play an indispensable role in this process by enhancing MIWI interaction with an APC/C substrate-binding subunit. Interestingly, piRNA-triggered MIWI destruction occurs in late spermatids, which in turn leads to piRNA elimination, suggesting a feedforward mechanism for coordinated removal of the MIWI/piRNA machinery at a specific developmental stage. Importantly, the proper removal of MIWI/piRNA is essential for sperm maturation. Together, our results reveal a role for piRNAs in regulating the clearance of the MIWI/piRNA machinery via the ubiquitin-proteosome pathway and demonstrate the critical importance of proper temporal regulation of MIWI/piRNA in male germ cell development.