RESUMO
Spermatogonial differentiation and meiotic initiation during spermatogenesis are tightly regulated by a number of genes, including those encoding enzymes for miRNA biogenesis. However, whether and how single miRNAs regulate these processes remain unclear. Here, we report that miR-202, a member of the let-7 family, prevents precocious spermatogonial differentiation and meiotic initiation in spermatogenesis by regulating the timely expression of many genes, including those for key regulators such as STRA8 and DMRT6. In miR-202 knockout (KO) mice, the undifferentiated spermatogonial pool is reduced, accompanied by age-dependent decline of fertility. In KO mice, SYCP3, STRA8 and DMRT6 are expressed earlier than in wild-type littermates, and Dmrt6 mRNA is a direct target of miR-202-5p. Moreover, the precocious spermatogonial differentiation and meiotic initiation were also observed in KO spermatogonial stem cells when cultured and induced in vitro, and could be partially rescued by the knockdown of Dmrt6. Therefore, we have not only shown that miR-202 is a regulator of meiotic initiation but also identified a previously unknown module in the underlying regulatory network.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , MicroRNAs/genética , Espermatogênese/genética , Espermatogônias/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Células-Tronco Germinativas Adultas/citologia , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Espermatogônias/metabolismo , Testículo/metabolismo , Fatores de Transcrição/genéticaRESUMO
MicroRNAs (miRNAs) are believed to play important roles in mammalian spermatogenesis but the in vivo functions of single miRNAs in this highly complex developmental process remain unclear. Here, we report that miR-202, a member of the let-7 family, plays an important role in spermatogenesis by phenotypic evaluation of miR-202 knockout (KO) mice. Loss of miR-202 results in spermatocyte apoptosis and perturbation of the zygonema-to-pachynema transition. Multiple processes during meiosis prophase I including synapsis and crossover formation are disrupted, and inter-sister chromatid synapses are detected. Moreover, we demonstrate that Separase mRNA is a miR-202 direct target and provides evidence that miR-202 upregulates REC8 by repressing Separase expression. Therefore, we have identified miR-202 as a new regulating noncoding gene that acts on the established SEPARASE-REC8 axis in meiosis.
Assuntos
Proteínas de Ciclo Celular , MicroRNAs , Separase , Animais , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Masculino , Meiose/genética , Camundongos , MicroRNAs/genética , Separase/genéticaRESUMO
Prime editor (PE) has tremendous promise for gene therapy. However, it remains a challenge to deliver PE (>6.3 kb) in vivo. Although PE can be split into two fragments and delivered using dual adeno-associated viruses (AAVs), choice of split sites within Cas9-which affects editing efficiency-is limited due to the large size of PE. Furthermore, overexpressing reverse transcriptase in mammalian cells might disrupt translation termination via its RNase H domain. Here, we developed a compact PE without the RNase H domain that showed editing comparable with full-length PE. With compact PE, we used a Cas9 split site (Glu 573) that supported robust editing in cells (up to 93% of full-length PE) and in mouse liver. We then demonstrated that split-cPE573 delivered by dual-AAV8 efficiently mediated a 3-bp TGA insertion in the Pcsk9 gene in mouse liver. Compact PE without the RNase H domain abolished its binding to peptidyl release factor 1 (eRF1) and mitigated the stop codon readthrough effect observed with full-length PE. This study identifies a compact PE with a flexible split design to advance utility of prime editing in vivo.
Assuntos
Edição de Genes , Pró-Proteína Convertase 9 , Animais , Fígado , Mamíferos , Camundongos , Pró-Proteína Convertase 9/genética , DNA Polimerase Dirigida por RNA , Ribonuclease HRESUMO
The postmeiotic development of male germ cells, also known as spermiogenesis, features the coordinated expression of a large number of spermatid-specific genes. However, only a limited number of key transcription factors have been identified and the underlying regulatory mechanisms remain largely unknown. Here, we report that SOX30, the most-divergent member of the Sry-related high-motility group box (SOX) family of transcription factors, is essential for mouse spermiogenesis. The SOX30 protein was predominantly expressed in spermatids, while its transcription was regulated by retinoic acid and by MYBL1 before and during meiosis. Sox30 knockout mice arrested spermiogenesis at step 3 round spermatids, which underwent apoptosis and abnormal chromocenter formation. We also determined that SOX30 regulated the expression of hundreds of spermatid-specific protein-coding and long non-coding RNA genes. SOX30 bound to the proximal promoter of its own gene and activated its transcription. These results reveal SOX30 as a novel key regulator of spermiogenesis that regulates its own transcription to enforce and activate this meiotic regulatory pathway.
Assuntos
Regulação da Expressão Gênica/genética , Fatores de Transcrição SOX/genética , Espermátides/metabolismo , Espermatogênese/fisiologia , Animais , Apoptose/fisiologia , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myb/genética , Transativadores/genética , Tretinoína/metabolismoRESUMO
Telomere attachment to the nuclear envelope (NE) is a prerequisite for chromosome movement during meiotic prophase I that is required for pairing of homologous chromosomes, synapsis, and homologous recombination. Here we show that Speedy A, a noncanonical activator of cyclin-dependent kinases (Cdks), is specifically localized to telomeres in prophase I male and female germ cells in mice, and plays an essential role in the telomere-NE attachment. Deletion of Spdya in mice disrupts telomere-NE attachment, and this impairs homologous pairing and synapsis and leads to zygotene arrest in male and female germ cells. In addition, we have identified a telomere localization domain on Speedy A covering the distal N terminus and the Cdk2-binding Ringo domain, and this domain is essential for the localization of Speedy A to telomeres. Furthermore, we found that the binding of Cdk2 to Speedy A is indispensable for Cdk2's localization on telomeres, suggesting that Speedy A and Cdk2 might be the initial components that are recruited to the NE for forming the meiotic telomere complex. However, Speedy A-Cdk2-mediated telomere-NE attachment is independent of Cdk2 activation. Our results thus indicate that Speedy A and Cdk2 might mediate the initial telomere-NE attachment for the efficient assembly of the telomere complex that is essential for meiotic prophase I progression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Quinase 2 Dependente de Ciclina/química , Ativação Enzimática , Feminino , Masculino , Prófase Meiótica I/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membrana Nuclear/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Espermatócitos/citologia , Espermatócitos/metabolismo , Telômero/metabolismoRESUMO
Family with sequence similarity 46, member C (FAM46C) is a highly conserved non-canonical RNA polyadenylation polymerase that is abundantly expressed in human and mouse testes and is frequently mutated in patients with multiple myeloma. However, its physiological role remains largely unknown. In this study, we found that FAM46C is specifically localized to the manchette of spermatids in mouse testes, a transient microtubule-based structure mainly involved in nuclear shaping and intra-flagellar protein traffic. Gene knockout of FAM46C in mice resulted in male sterility, characterized by the production of headless spermatozoa in testes. Sperm heads were intermittently found in the epididymides of FAM46C knockout mice, but their fertilization ability was severely compromised based on the results of intracytoplasmic sperm injection assays. Interestingly, our RNA-sequencing analyses of FAM46C knockout testes revealed that mRNA levels of only nine genes were significantly altered compared to wild-type ones (q < 0.05). When considering alternate activities for FAM46C, in vitro assays demonstrated that FAM46C does not exhibit protein kinase or AMPylation activity against general substrates. Together, our data show that FAM46C in spermatids is a novel component in fastening the sperm head and flagellum.
Assuntos
Flagelos/fisiologia , Polinucleotídeo Adenililtransferase/fisiologia , Cabeça do Espermatozoide/fisiologia , Espermátides/fisiologia , Espermatogênese/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Feminino , Flagelos/metabolismo , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polinucleotídeo Adenililtransferase/genética , Gravidez , Cabeça do Espermatozoide/metabolismo , Espermátides/citologia , Espermatozoides/fisiologiaRESUMO
miRNAs play important roles during mammalian spermatogenesis. However, the function of most miRNAs in spermatogenesis and the underlying mechanisms remain unknown. Here, we report that miR-202 is highly expressed in mouse spermatogonial stem cells (SSCs), and is oppositely regulated by Glial cell-Derived Neurotrophic Factor (GDNF) and retinoic acid (RA), two key factors for SSC self-renewal and differentiation. We used inducible CRISPR-Cas9 to knockout miR-202 in cultured SSCs, and found that the knockout SSCs initiated premature differentiation accompanied by reduced stem cell activity and increased mitosis and apoptosis. Target genes were identified with iTRAQ-based proteomic analysis and RNA sequencing, and are enriched with cell cycle regulators and RNA-binding proteins. Rbfox2 and Cpeb1 were found to be direct targets of miR-202 and Rbfox2 but not Cpeb1, is essential for the differentiation of SSCs into meiotic cells. Accordingly, an SSC fate-regulatory network composed of signaling molecules of GDNF and RA, miR-202 and diverse downstream effectors has been identified.
Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Ciclo Celular/genética , MicroRNAs/metabolismo , Fatores de Processamento de RNA/biossíntese , Células-Tronco Germinativas Adultas/citologia , Animais , Técnicas de Inativação de Genes , Masculino , Meiose/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Proteômica , Análise de Sequência de RNA , Espermatogênese/genética , Fatores de Transcrição/biossíntese , Fatores de Poliadenilação e Clivagem de mRNA/biossínteseRESUMO
Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function. We show that CK2 is required for the localization of PRG-1 and for the proper localization of several factors that comprise the 'upstream sequence transcription complex' (USTC), which is required for piRNA transcription. Loss of CK2 impairs piRNA levels suggesting that CK2 promotes USTC function. We identify the USTC component twenty-one-U fouled-up 4 (TOFU-4) as a direct substrate for CK2. Our findings suggest that phosphorylation of TOFU-4 by CK2 promotes the assembly of USTC and piRNA transcription. Notably, during the aging process, CK2 activity declines, resulting in the disassembly of USTC, decreased piRNA production, and defects in piRNA-mediated gene silencing, including transposons silencing. These findings highlight the significance of posttranslational modification in regulating piRNA biogenesis and its implications for the aging process. Overall, our study provides compelling evidence for the involvement of a posttranslational modification mechanism in the regulation of piRNA biogenesis.
Assuntos
Proteínas de Drosophila , Alimentos de Soja , Animais , RNA de Interação com Piwi , RNA Interferente Pequeno/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Fosforilação , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genéticaRESUMO
Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function. We show that CK2 is required for the localization of PRG-1 and for the proper localization of several factors that comprise the 'upstream sequence transcription complex' (USTC), which is required for piRNA transcription. Loss of CK2 impairs piRNA levels suggesting that CK2 promotes USTC function. We identify the USTC component twenty-one-U fouled-up 4 (TOFU-4) as a direct substrate for CK2. Our findings suggest that phosphorylation of TOFU-4 by CK2 promotes the assembly of USTC and piRNA transcription. Notably, during the aging process, CK2 activity declines, resulting in the disassembly of USTC, decreased piRNA production, and defects in piRNA-mediated gene silencing, including transposons silencing. These findings highlight the significance of posttranslational modification in regulating piRNA biogenesis and its implications for the aging process. Overall, our study provides compelling evidence for the involvement of a posttranslational modification mechanism in the regulation of piRNA biogenesis.
RESUMO
Targeted insertion of large DNA fragments holds promise for genome engineering and gene therapy. Prime editing (PE) effectively inserts short (<50 bp) sequences. Employing paired prime editing guide RNAs (pegRNAs) has enabled PE to better mediate relatively large insertions in vitro, but the efficiency of larger insertions (>400 bp) remains low and in vivo application has not been demonstrated. Inspired by the efficient genomic insertion mechanism of retrotransposons, we develop a template-jumping (TJ) PE approach for the insertion of large DNA fragments using a single pegRNA. TJ-pegRNA harbors the insertion sequence as well as two primer binding sites (PBSs), with one PBS matching a nicking sgRNA site. TJ-PE precisely inserts 200 bp and 500 bp fragments with up to 50.5 and 11.4% efficiency, respectively, and enables GFP (~800 bp) insertion and expression in cells. We transcribe split circular TJ-petRNA in vitro via a permuted group I catalytic intron for non-viral delivery in cells. Finally, we demonstrate that TJ-PE can rewrite an exon in the liver of tyrosinemia I mice to reverse the disease phenotype. TJ-PE has the potential to insert large DNA fragments without double-stranded DNA breaks and facilitate mutation hotspot exon rewriting in vivo.
Assuntos
DNA , Edição de Genes , Camundongos , Animais , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Éxons/genética , Genoma , Sistemas CRISPR-Cas/genéticaRESUMO
Reverse transcriptases, used in prime editing systems, exhibit lower fidelity, processivity and dNTP affinity than many DNA-dependent DNA polymerases. We report that a DNA-dependent DNA polymerase (phi29), untethered from Cas9, enables editing from a synthetic, end-stabilized DNA-containing template at up to 60% efficiency in human cells. Compared to prime editing, DNA polymerase editing avoids autoinhibitory intramolecular base pairing of the template, facilitates template synthesis and supports larger insertions (>100 nucleotides).
RESUMO
Delivery and optimization of prime editors (PEs) have been hampered by their large size and complexity. Although split versions of genome-editing tools can reduce construct size, they require special engineering to tether the binding and catalytic domains. Here we report a split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT). The sPE showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion-deletion (indel) byproducts. Delivery of sPE to the mouse liver with hydrodynamic injection to modify ß-catenin drove tumor formation with similar efficiency as PE3. Delivery with two adeno-associated virus (AAV) vectors corrected the disease-causing mutation in a mouse model of type I tyrosinemia. Similarly, prime editing guide RNAs (pegRNAs) can be split into a single guide RNA (sgRNA) and a circular RNA RT template to increase flexibility and stability. Compared to previous sPEs, ours lacks inteins, protein-protein affinity modules and nuclease-sensitive pegRNA extensions, which increase construct complexity and might reduce efficiency. Our modular system will facilitate the delivery and optimization of PEs.
Assuntos
RNA Circular , Tirosinemias , Animais , Sistemas CRISPR-Cas , Desoxirribonuclease I/genética , Edição de Genes , Camundongos , RNA Circular/genética , RNA Guia de Cinetoplastídeos/genética , DNA Polimerase Dirigida por RNA/genética , Tirosinemias/genéticaRESUMO
Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.
Assuntos
Carcinogênese/genética , Edição de Genes/métodos , Neoplasias/genética , RNA Guia de Cinetoplastídeos/genética , Alelos , Animais , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Modelos Animais de Doenças , Células HEK293 , Células HeLa , Humanos , Camundongos , Neoplasias/patologia , TransfecçãoRESUMO
Cultured mouse spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), revert back to pluripotent state either spontaneously or upon being modified genetically. However, the reprogramming efficiencies are low, and the underlying mechanism remains poorly understood. In the present study, we conducted transcriptomic analysis and found that many transcription factors and epigenetic modifiers were differentially expressed between GSCs and embryonic stem cells. We failed in reprogramming GSCs to pluripotent state using the Yamanaka 4 Factors, but succeeded when Nanog and Tet1 were included. More importantly, reprogramming was also achieved with Nanog alone in a p53-deficient GSC line with an efficiency of 0.02. These GSC-derived-induced pluripotent stem cells possessed in vitro and in vivo differentiation abilities despite the low rate of chimera formation, which might be caused by abnormal methylation in certain paternally imprinted genes. Together, these results show that GSCs can be reprogrammed to pluripotent state via multiple avenues and contribute to our understanding of the mechanisms of GSC reprogramming.
Assuntos
Reprogramação Celular/fisiologia , Proteína Homeobox Nanog/metabolismo , Células-Tronco Pluripotentes/metabolismo , Espermatogônias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Metilação de DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Perfilação da Expressão Gênica/métodos , Masculino , Camundongos , Espermatogônias/fisiologia , Fatores de Transcrição/metabolismoRESUMO
ZMYM3, a member of the MYM-type zinc finger protein family and a component of a LSD1-containing transcription repressor complex, is predominantly expressed in the mouse brain and testis. Here, we show that ZMYM3 in the mouse testis is expressed in somatic cells and germ cells until pachytene spermatocytes. Knockout (KO) of Zmym3 in mice using the CRISPR-Cas9 system resulted in adult male infertility. Spermatogenesis of the KO mice was arrested at the metaphase of the first meiotic division (MI). ZMYM3 co-immunoprecipitated with LSD1 in spermatogonial stem cells, but its KO did not change the levels of LSD1 or H3K4me1/2 or H3K9me2. However, Zmym3 KO resulted in elevated numbers of apoptotic germ cells and of MI spermatocytes that are positive for BUB3, which is a key player in spindle assembly checkpoint. Zmym3 KO also resulted in up-regulated expression of meiotic genes in spermatogonia. These results show that ZMYM3 has an essential role in metaphase to anaphase transition during mouse spermatogenesis by regulating the expression of diverse families of genes.
Assuntos
Meiose/genética , Proteínas Nucleares/genética , Espermatogênese/genética , Testículo/crescimento & desenvolvimento , Animais , Pontos de Checagem da Fase M do Ciclo Celular/genética , Masculino , Metáfase/genética , Camundongos , Camundongos Knockout , Espermatócitos/crescimento & desenvolvimento , Testículo/metabolismoRESUMO
Meiosis is the key step in gametogenesis. However, the mechanism of mammalian meiosis remains poorly understood due to the lack of an in vitro model. Here, we report that retinoic acid (RA) is sufficient for inducing leptotene/zygotene spermatocytes from cultured mouse spermatogonial stem cells. Multiple genes regulated by RA were identified by RNA sequencing. RA in combination with pup Sertoli cell co-culture resulted in a higher induction efficiency of 28%. Comparisons in the transcriptomic profiles of the induced spermatogenic cells and the isolated ones revealed the progressive induction of the germ cells. Using this model, we showed that Stra8, Agpat3, Fam57a, Wdr91, and Sox30 contributed to the proliferation and meiosis initiation differentially. In conclusion, we have efficiently generated spermatocytes using an RA/pup Sertoli cell-based in vitro model and provided proof-of-concept evidence for its application in identifying genes involved in mammalian meiosis.