RESUMEN
piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant. These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells.
Asunto(s)
Elementos Transponibles de ADN , Infertilidad Masculina , ARN Interferente Pequeño , Espermatogénesis , Testículo , Masculino , Humanos , Espermatogénesis/genética , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/genética , Elementos Transponibles de ADN/genética , Animales , Testículo/metabolismo , Ratones , Adulto , Silenciador del Gen , Ratones Noqueados , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Elementos de Nucleótido Esparcido Largo/genética , Espermatogonias/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ARN de Interacción con PiwiRESUMEN
Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.
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Progresión de la Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Leucemia Mieloide Aguda , Inhibidores de Prolil-Hidroxilasa , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Inhibidores de Prolil-Hidroxilasa/farmacología , Inhibidores de Prolil-Hidroxilasa/uso terapéutico , Animales , Ratones , Apoptosis/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Línea Celular Tumoral , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Estabilidad Proteica/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con PuentesRESUMEN
N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. The loss of TDRD5 and TDRKH interaction with MIWI results in attenuation of piRNA amplification. We find that piRNA amplification is necessary for transposon control and for sustaining piRNA levels including select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as self-serving genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.
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Arginina , Proteínas Argonautas , Fase Paquiteno , ARN Interferente Pequeño , Espermatogénesis , Animales , Masculino , Ratones , Arginina/metabolismo , Arginina/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Elementos Transponibles de ADN , ARN de Interacción con Piwi , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Dominio TudorRESUMEN
In the male mouse germ line, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germ line remain unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of these pathogenic alleles, we discovered that the uncharacterized protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in fetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterized protein essential for orchestrating piRNA-directed DNA methylation.
Asunto(s)
Metilación de ADN , ARN de Interacción con Piwi , Masculino , Humanos , Animales , Ratones , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas/metabolismo , Células Germinativas/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Elementos Transponibles de ADN/genética , Mamíferos/metabolismoRESUMEN
The paradigmatic hematopoietic tree model is increasingly recognized to be limited, as it is based on heterogeneous populations largely defined by non-homeostatic assays testing cell fate potentials. Here, we combine persistent labeling with time-series single-cell RNA sequencing to build a real-time, quantitative model of in vivo tissue dynamics for murine bone marrow hematopoiesis. We couple cascading single-cell expression patterns with dynamic changes in differentiation and growth speeds. The resulting explicit linkage between molecular states and cellular behavior reveals widely varying self-renewal and differentiation properties across distinct lineages. Transplanted stem cells show strong acceleration of differentiation at specific stages of erythroid and neutrophil production, illustrating how the model can quantify the impact of perturbations. Our reconstruction of dynamic behavior from snapshot measurements is akin to how a kinetoscope allows sequential images to merge into a movie. We posit that this approach is generally applicable to understanding tissue-scale dynamics at high resolution.
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Médula Ósea , Células Madre Hematopoyéticas , Animales , Ratones , Células Madre Hematopoyéticas/metabolismo , Hematopoyesis/genética , Diferenciación CelularRESUMEN
N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. Surprisingly, the loss of TDRD5 and TDRKH interaction with MIWI results in defective piRNA amplification, rather than an expected failure of piRNA biogenesis. We find that piRNA amplification is necessary for both transposon control and for sustaining levels of select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as autonomous genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.
RESUMEN
RNA-directed transposon silencing operates in the mammalian soma and germline to safeguard genomic integrity. The piRNA pathway and the HUSH complex identify active transposons through recognition of their nascent transcripts, but mechanistic understanding of how these distinct pathways evolved is lacking. TASOR is an essential component of the HUSH complex. TASOR's DUF3715 domain adopts a pseudo-PARP structure and is required for transposon silencing in a manner independent of complex assembly. TEX15, an essential piRNA pathway factor, also contains the DUF3715 domain. Here, we show that TASOR's and TEX15's DUF3715 domain share extensive structural homology. We found that the DUF3715 domain arose in early eukaryotes and that in vertebrates it is restricted to TEX15, TASOR, and TASORB orthologs. While TASOR-like proteins are found throughout metazoa, TEX15 is vertebrate-specific. The branching of TEX15 and the TASOR-like DUF3715 domain likely occurred in early metazoan evolution. Remarkably, despite this vast evolutionary distance, the DUF3715 domain from divergent TEX15 sequences can functionally substitute the DUF3715 domain of TASOR and mediates transposon silencing. We have thus termed this domain of unknown function as the RNA-directed pseudo-PARP transposon silencing (RDTS) domain. In summary, we show an unexpected functional link between these critical transposon silencing pathways.
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Proteínas de Drosophila , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Animales , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Interferencia de ARN , Genoma , Proteínas Argonautas/genética , ARN de Interacción con Piwi , Mamíferos/genética , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genéticaRESUMEN
MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicerâ¢-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.
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MicroARNs , Ribonucleasa III , Ratones , Animales , Ribonucleasa III/metabolismo , Interferencia de ARN , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Portadoras/metabolismo , Mamíferos/metabolismoRESUMEN
Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic differentiation hierarchy and sustain multilineage hematopoiesis. Here, we show that the transcriptional regulator CITED2 is essential for life-long HSC maintenance. While hematopoietic-specific Cited2 deletion has a minor impact on steady-state hematopoiesis, Cited2-deficient HSCs are severely depleted in young mice and fail to expand upon aging. Moreover, although they home normally to the bone marrow, they fail to reconstitute hematopoiesis upon transplantation. Mechanistically, CITED2 is required for expression of key HSC regulators, including GATA2, MCL-1, and PTEN. Hematopoietic-specific expression of anti-apoptotic MCL-1 partially rescues the Cited2-deficient HSC pool and restores their reconstitution potential. To interrogate the Cited2âPten pathway in HSCs, we generated Cited2;Pten compound heterozygous mice, which had a decreased number of HSCs that failed to reconstitute the HSC compartment. In addition, CITED2 represses multiple pathways whose elevated activity causes HSC exhaustion. Thus, CITED2 promotes pathways necessary for HSC maintenance and suppresses those detrimental to HSC integrity.
Asunto(s)
Regulación de la Expresión Génica , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/metabolismo , Proteínas Represoras/genética , Transactivadores/genética , Animales , Apoptosis/genética , Proliferación Celular/genética , Redes Reguladoras de Genes/genética , Ratones Endogámicos C57BL , Ratones Noqueados , RNA-Seq/métodos , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Factores de Tiempo , Transactivadores/metabolismoRESUMEN
Spermatogonial stem cells (SSCs) sustain spermatogenesis and fertility throughout adult male life. The conserved RNA-binding protein NANOS2 is essential for the maintenance of SSCs, but its targets and mechanisms of function are not fully understood. Here, we generated a fully functional epitope-tagged Nanos2 mouse allele and applied the highly stringent cross-linking and analysis of cDNAs to define NANOS2 RNA occupancy in SSC lines. NANOS2 recognizes the AUKAAWU consensus motif, mostly found in the 3' untranslated region of defined messenger RNAs (mRNAs). We find that NANOS2 is a regulator of key signaling and metabolic pathways whose dosage or activity are known to be critical for SSC maintenance. NANOS2 interacts with components of CCR4-NOT deadenylase complex in SSC lines, and consequently, NANOS2 binding reduces the half-lives of target transcripts. In summary, NANOS2 contributes to SSC maintenance through the regulation of target mRNA stability and key self-renewal pathways.
RESUMEN
Lifelong multilineage hematopoiesis critically depends on rare hematopoietic stem cells (HSCs) that reside in the hypoxic bone marrow microenvironment. Although the role of the canonical oxygen sensor hypoxia-inducible factor prolyl hydroxylase has been investigated extensively in hematopoiesis, the functional significance of other members of the 2-oxoglutarate (2-OG)-dependent protein hydroxylase family of enzymes remains poorly defined in HSC biology and multilineage hematopoiesis. Here, by using hematopoietic-specific conditional gene deletion, we reveal that the 2-OG-dependent protein hydroxylase JMJD6 is essential for short- and long-term maintenance of the HSC pool and multilineage hematopoiesis. Additionally, upon hematopoietic injury, Jmjd6-deficient HSCs display a striking failure to expand and regenerate the hematopoietic system. Moreover, HSCs lacking Jmjd6 lose multilineage reconstitution potential and self-renewal capacity upon serial transplantation. At the molecular level, we found that JMJD6 functions to repress multiple processes whose downregulation is essential for HSC integrity, including mitochondrial oxidative phosphorylation (OXPHOS), protein synthesis, p53 stabilization, cell cycle checkpoint progression, and mTORC1 signaling. Indeed, Jmjd6-deficient primitive hematopoietic cells display elevated basal and maximal mitochondrial respiration rates and increased reactive oxygen species (ROS), prerequisites for HSC failure. Notably, an antioxidant, N-acetyl-l-cysteine, rescued HSC and lymphoid progenitor cell depletion, indicating a causal impact of OXPHOS-mediated ROS generation upon Jmjd6 deletion. Thus, JMJD6 promotes HSC maintenance and multilineage differentiation potential by suppressing fundamental pathways whose activation is detrimental for HSC function.
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Hematopoyesis , Células Madre Hematopoyéticas , Médula Ósea , Trasplante de Médula Ósea , Diferenciación CelularRESUMEN
[Figure: see text].
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Aterosclerosis/etiología , Desdiferenciación Celular , MicroARNs/metabolismo , Músculo Liso Vascular/fisiología , ARN Largo no Codificante/fisiología , Animales , Aterosclerosis/patología , Movimiento Celular , Proliferación Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Vasos Coronarios/citología , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Metabolismo de los Lípidos , Ratones , Músculo Liso Vascular/citología , Oligonucleótidos Antisentido , Fenotipo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , TranscriptomaRESUMEN
The mRNA N6-methyladenosine (m6A) modification has emerged as an essential regulator of normal and malignant hematopoiesis. Inactivation of the m6A mRNA reader YTHDF2, which recognizes m6A-modified transcripts to promote m6A-mRNA degradation, results in hematopoietic stem cell (HSC) expansion and compromises acute myeloid leukemia. Here we investigate the long-term impact of YTHDF2 deletion on HSC maintenance and multilineage hematopoiesis. We demonstrate that Ythdf2-deficient HSCs from young mice fail upon serial transplantation, display increased abundance of multiple m6A-modified inflammation-related transcripts, and chronically activate proinflammatory pathways. Consistent with the detrimental consequences of chronic activation of inflammatory pathways in HSCs, hematopoiesis-specific Ythdf2 deficiency results in a progressive myeloid bias, loss of lymphoid potential, HSC expansion, and failure of aged Ythdf2-deficient HSCs to reconstitute multilineage hematopoiesis. Experimentally induced inflammation increases YTHDF2 expression, and YTHDF2 is required to protect HSCs from this insult. Thus, our study positions YTHDF2 as a repressor of inflammatory pathways in HSCs and highlights the significance of m6A in long-term HSC maintenance.
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Adenosina/análogos & derivados , Células Madre Hematopoyéticas/metabolismo , Inflamación/genética , Proteínas de Unión al ARN/metabolismo , Adenosina/metabolismo , Animales , Linaje de la Célula , Proliferación Celular , Senescencia Celular , Eliminación de Gen , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Inflamación/patología , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation1. In the male germline, RNA-directed DNA methylation silences young, active transposable elements2-4. The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements3,5. piRNAs are proposed to tether MIWI2 to nascent transposable element transcripts; however, the mechanism by which MIWI2 directs the de novo methylation of transposable elements is poorly understood, although central to the immortality of the germline. Here we define the interactome of MIWI2 in mouse fetal gonocytes undergoing de novo genome methylation and identify a previously unknown MIWI2-associated factor, SPOCD1, that is essential for the methylation and silencing of young transposable elements. The loss of Spocd1 in mice results in male-specific infertility but does not affect either piRNA biogenesis or the localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein whose expression is restricted to the period of de novo genome methylation. It co-purifies in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery, as well as with constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent transposable element transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through SPOCD1. In summary, we have identified a previously unrecognized and essential executor of mammalian piRNA-directed DNA methylation.
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Metilación de ADN/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/metabolismo , Ensamble y Desensamble de Cromatina , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Elementos Transponibles de ADN/genética , Femenino , Fertilidad/genética , Silenciador del Gen , Genes de Partícula A Intracisternal/genética , Elementos de Nucleótido Esparcido Largo/genética , Masculino , Ratones , ARN Interferente Pequeño/biosíntesis , Espermatogénesis/genéticaRESUMEN
The PIWI protein MIWI2 and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of young active transposable elements (TEs) in the male germline. piRNAs are proposed to recruit MIWI2 to the transcriptionally active TE loci by base pairing to nascent transcripts, however the downstream mechanisms and effector proteins utilized by MIWI2 in directing de novo TE methylation remain incompletely understood. Here, we show that MIWI2 associates with TEX15 in foetal gonocytes. TEX15 is predominantly a nuclear protein that is not required for piRNA biogenesis but is essential for piRNA-directed TE de novo methylation and silencing. In summary, TEX15 is an essential executor of mammalian piRNA-directed DNA methylation.
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Proteínas Argonautas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Metilación de ADN/genética , Elementos Transponibles de ADN/genética , Silenciador del Gen , Animales , Proteínas Argonautas/genética , Femenino , Feto/citología , Genoma , Células Germinativas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Testículo/metabolismoRESUMEN
Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion.
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Leucemia Mieloide Aguda/terapia , Células Madre Neoplásicas/fisiología , Proteínas de Unión al ARN/metabolismo , Animales , Autorrenovación de las Células , Hematopoyesis , Células Madre Hematopoyéticas , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Células THP-1RESUMEN
Several developmental stages of spermatogenesis are transcriptionally quiescent which presents major challenges associated with the regulation of gene expression. Here we identify that the zygotene to pachytene transition is not only associated with the resumption of transcription but also a wave of programmed mRNA degradation that is essential for meiotic progression. We explored whether terminal uridydyl transferase 4- (TUT4-) or TUT7-mediated 3' mRNA uridylation contributes to this wave of mRNA degradation during pachynema. Indeed, both TUT4 and TUT7 are expressed throughout most of spermatogenesis, however, loss of either TUT4 or TUT7 does not have any major impact upon spermatogenesis. Combined TUT4 and TUT7 (TUT4/7) deficiency results in embryonic growth defects, while conditional gene targeting revealed an essential role for TUT4/7 in pachytene progression. Loss of TUT4/7 results in the reduction of miRNA, piRNA and mRNA 3' uridylation. Although this reduction does not greatly alter miRNA or piRNA expression, TUT4/7-mediated uridylation is required for the clearance of many zygotene-expressed transcripts in pachytene cells. We find that TUT4/7-regulated transcripts in pachytene spermatocytes are characterized by having long 3' UTRs with length-adjusted enrichment for AU-rich elements. We also observed these features in TUT4/7-regulated maternal transcripts whose dosage was recently shown to be essential for sculpting a functional maternal transcriptome and meiosis. Therefore, mRNA 3' uridylation is a critical determinant of both male and female germline transcriptomes. In conclusion, we have identified a novel requirement for 3' uridylation-programmed zygotene mRNA clearance in pachytene spermatocytes that is essential for male meiotic progression.
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Profase Meiótica I/genética , Fase Paquiteno/genética , Procesamiento Postranscripcional del ARN/fisiología , Espermatogénesis/genética , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Estabilidad del ARN/genética , ARN Mensajero/genética , UDP-Glucosa-Hexosa-1-Fosfato Uridiltransferasa/metabolismoRESUMEN
In animals, PIWI-interacting RNAs (piRNAs) of 21-35 nucleotides in length silence transposable elements, regulate gene expression and fight viral infection. piRNAs guide PIWI proteins to cleave target RNA, promote heterochromatin assembly and methylate DNA. The architecture of the piRNA pathway allows it both to provide adaptive, sequence-based immunity to rapidly evolving viruses and transposons and to regulate conserved host genes. piRNAs silence transposons in the germ line of most animals, whereas somatic piRNA functions have been lost, gained and lost again across evolution. Moreover, most piRNA pathway proteins are deeply conserved, but different animals employ remarkably divergent strategies to produce piRNA precursor transcripts. Here, we discuss how a common piRNA pathway allows animals to recognize diverse targets, ranging from selfish genetic elements to genes essential for gametogenesis.
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Elementos Transponibles de ADN , Evolución Molecular , Silenciador del Gen , ARN Interferente Pequeño , Virosis , Virus , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Virosis/genética , Virosis/metabolismo , Virus/genética , Virus/metabolismoRESUMEN
RNA viruses are a major threat to animals and plants. RNA interference (RNAi) and the interferon response provide innate antiviral defense against RNA viruses. Here, we performed a large-scale screen using Caenorhabditis elegans and its natural pathogen the Orsay virus (OrV), and we identified cde-1 as important for antiviral defense. CDE-1 is a homolog of the mammalian TUT4 and TUT7 terminal uridylyltransferases (collectively called TUT4(7)); its catalytic activity is required for its antiviral function. CDE-1 uridylates the 3' end of the OrV RNA genome and promotes its degradation in a manner independent of the RNAi pathway. Likewise, TUT4(7) enzymes uridylate influenza A virus (IAV) mRNAs in mammalian cells. Deletion of TUT4(7) leads to increased IAV mRNA and protein levels. Collectively, these data implicate 3'-terminal uridylation of viral RNAs as a conserved antiviral defense mechanism.
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Caenorhabditis elegans/enzimología , Caenorhabditis elegans/virología , Inmunidad Innata , ARN Nucleotidiltransferasas/metabolismo , Virus ARN/metabolismo , Células A549 , Animales , Caenorhabditis elegans/genética , Humanos , Interferencia de ARN , Virus ARN/inmunología , Virus ARN/fisiología , ARN Viral/metabolismo , Transcriptoma , Replicación ViralRESUMEN
Defective germline reprogramming in Piwil4 (Miwi2)- and Dnmt3l-deficient mice results in the failure to reestablish transposon silencing, meiotic arrest and progressive loss of spermatogonia. Here we sought to understand the molecular basis for this spermatogonial dysfunction. Through a combination of imaging, conditional genetics and transcriptome analysis, we demonstrate that germ cell elimination in the respective mutants arises as a result of defective de novo genome methylation during reprogramming rather than because of a function for the respective factors within spermatogonia. In both Miwi2-/- and Dnmt3l-/- spermatogonia, the intracisternal-A particle (IAP) family of endogenous retroviruses is derepressed, but, in contrast to meiotic cells, DNA damage is not observed. Instead, we find that unmethylated IAP promoters rewire the spermatogonial transcriptome by driving expression of neighboring genes. Finally, spermatogonial numbers, proliferation and differentiation are altered in Miwi2-/- and Dnmt3l-/- mice. In summary, defective reprogramming deregulates the spermatogonial transcriptome and may underlie spermatogonial dysfunction.