RESUMEN
Complex diseases often involve the interplay between genetic and environmental factors. Charcot-Marie-Tooth type 2 neuropathies (CMT2) are a group of genetically heterogeneous disorders, in which similar peripheral neuropathology is inexplicably caused by various mutated genes. Their possible molecular links remain elusive. Here, we found that upon environmental stress, many CMT2-causing mutant proteins adopt similar properties by entering stress granules (SGs), where they aberrantly interact with G3BP and integrate into SG pathways. For example, glycyl-tRNA synthetase (GlyRS) is translocated from the cytoplasm into SGs upon stress, where the mutant GlyRS perturbs the G3BP-centric SG network by aberrantly binding to G3BP. This disrupts SG-mediated stress responses, leading to increased stress vulnerability in motoneurons. Disrupting this aberrant interaction rescues SG abnormalities and alleviates motor deficits in CMT2D mice. These findings reveal a stress-dependent molecular link across diverse CMT2 mutants and provide a conceptual framework for understanding genetic heterogeneity in light of environmental stress.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Animales , Ratones , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Citoplasma , Neuronas Motoras , Proteínas con Motivos de Reconocimiento de ARN/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.
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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
The translocation of stimulator of interferon genes (STING) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) enables its activation. However, the mechanism underlying the regulation of STING exit from the ER remains elusive. Here, we found that STING induces the activation of transforming growth factor beta-activated kinase 1 (TAK1) prior to STING trafficking in a TAK1 binding protein 1 (TAB1)-dependent manner. Intriguingly, activated TAK1 directly mediates STING phosphorylation on serine 355, which facilitates its interaction with STING ER exit protein (STEEP) and thereby promotes its oligomerization and translocation to the ERGIC for subsequent activation. Importantly, activation of TAK1 by monophosphoryl lipid A, a TLR4 agonist, boosts cGAMP-induced antitumor immunity dependent on STING phosphorylation in a mouse allograft tumor model. Taken together, TAK1 was identified as a checkpoint for STING activation by promoting its trafficking, providing a basis for combinatory tumor immunotherapy and intervention in STING-related diseases.
Asunto(s)
Neoplasias , Animales , Ratones , FosforilaciónRESUMEN
Virus infection modulates both host immunity and host genomic stability. Poly(ADP-ribose) polymerase 1 (PARP1) is a key nuclear sensor of DNA damage, which maintains genomic integrity, and the successful application of PARP1 inhibitors for clinical anti-cancer therapy has lasted for decades. However, precisely how PARP1 gains access to cytoplasm and regulates antiviral immunity remains unknown. Here, we report that DNA virus induces a reactive nitrogen species (RNS)-dependent DNA damage and activates DNA-dependent protein kinase (DNA-PK). Activated DNA-PK phosphorylates PARP1 on Thr594, thus facilitating the cytoplasmic translocation of PARP1 to inhibit the antiviral immunity both in vitro and in vivo. Mechanistically, cytoplasmic PARP1 interacts with and directly PARylates cyclic GMP-AMP synthase (cGAS) on Asp191 to inhibit its DNA-binding ability. Together, our findings uncover an essential role of PARP1 in linking virus-induced genome instability with inhibition of host immunity, which is of relevance to cancer, autoinflammation, and other diseases.
Asunto(s)
Antivirales , Nucleotidiltransferasas , Antivirales/farmacología , Citoplasma/genética , Citoplasma/metabolismo , ADN , Daño del ADN , Inestabilidad Genómica , Humanos , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismoRESUMEN
The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.
Asunto(s)
Nucléolo Celular , Exosomas , Precursores del ARN , Procesamiento Postranscripcional del ARN , ARN Ribosómico , Pez Cebra , Animales , Ratones , Nucléolo Celular/metabolismo , Desarrollo Embrionario , Exosomas/metabolismo , Cabeza/anomalías , Microscopía , Proteínas Nucleares/metabolismo , Precursores del ARN/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Ribosómico 28S/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
U6 snRNA, as an essential component of the catalytic core of the pre-mRNA processing spliceosome, is heavily modified post-transcriptionally, with 2'-O-methylation being most common. The role of these modifications in pre-mRNA splicing as well as their physiological function in mammals have remained largely unclear. Here we report that the La-related protein LARP7 functions as a critical cofactor for 2'-O-methylation of U6 in mouse male germ cells. Mechanistically, LARP7 promotes U6 loading onto box C/D snoRNP, facilitating U6 2'-O-methylation by box C/D snoRNP. Importantly, ablation of LARP7 in the male germline causes defective U6 2'-O-methylation, massive alterations in pre-mRNA splicing, and spermatogenic failure in mice, which can be rescued by ectopic expression of wild-type LARP7 but not an U6-loading-deficient mutant LARP7. Our data uncover a novel role of LARP7 in regulating U6 2'-O-methylation and demonstrate the functional requirement of such modification for splicing fidelity and spermatogenesis in mice.
Asunto(s)
Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Espermatogénesis , Espermatozoides/metabolismo , Empalmosomas/metabolismo , Animales , Fertilidad , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Masculino , Metilación , Ratones Endogámicos C57BL , Ratones Noqueados , Precursores del ARN/genética , ARN Mensajero/genética , ARN Nuclear Pequeño/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Transducción de Señal , Espermatogénesis/genética , Empalmosomas/genéticaRESUMEN
Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA-binding protein Rbm14 and maternal mRNAs co-phase separate into cytoplasmic condensates to facilitate vertebrate blastula-to-gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal γ-tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8- and 16-cell embryos. Their formation was dose-dependently stimulated by m6 A, but repressed by m5 C modification of the maternal mRNA. Furthermore, deadenylase Parn co-phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m6 A-modified maternal mRNAs, and by releasing the poly(A)-less transcripts upon regulated disassembly to allow their re-polyadenylation and translation or clearance.
Asunto(s)
ARN Mensajero Almacenado , Pez Cebra , Animales , Femenino , Ratones , Embarazo , Blastocisto/metabolismo , Blástula/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mensajero Almacenado/genética , ARN Mensajero Almacenado/metabolismoRESUMEN
Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.
Asunto(s)
Células Madre Embrionarias/citología , Técnicas Genéticas , Ratones Transgénicos , Animales , Blastocisto/citología , Núcleo Celular/metabolismo , Femenino , Marcación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Oocitos/citología , Oocitos/metabolismoRESUMEN
The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.
Asunto(s)
Exosomas/genética , Exosomas/metabolismo , Proteínas Nucleares/fisiología , ARN Helicasas/metabolismo , Animales , Núcleo Celular/metabolismo , Células Madre Embrionarias , Células HEK293 , Células HeLa , Humanos , Ratones , Proteínas Nucleares/genética , Unión Proteica , Dominios Proteicos , Transporte de Proteínas/genética , Estabilidad del ARN/genéticaRESUMEN
Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by Tars2), disruption of which accumulated Ser-tRNAThr and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.
Asunto(s)
Aminoacil-ARNt Sintetasas , Cardiomiopatías , Cardiopatías , Animales , Ratones , Especies Reactivas de Oxígeno , Puntos de Control del Ciclo Celular , Estrés Oxidativo , MamíferosRESUMEN
The second heart field (SHF) plays a pivotal role in heart development, particularly in outflow tract (OFT) morphogenesis and septation, as well as in the expansion of the right ventricle (RV). Two mouse Cre lines, the Mef2c-AHF-Cre (Mef2c-Cre) and Isl1-Cre, have been widely used to study the SHF development. However, Cre activity is triggered not only in the SHF but also in the RV in the Mef2c-Cre mice, and in the Isl1-Cre mice, Cre activation is not SHF-specific. Therefore, a more suitable SHF-Cre line is desirable for better understanding SHF development. Here, we generated and characterized the Prdm1-Cre knock-in mice. In comparison with Mef2c-Cre mice, the Cre activity is similar in the pharyngeal and splanchnic mesoderm, and in the OFT of the Prdm1-Cre mice. Nonetheless, it was noticed that Cre expression is largely reduced in the RV of Prdm1-Cre mice compared to the Mef2c-Cre mice. Furthermore, we deleted Hand2, Nkx2-5, Pdk1 and Tbx20 using both Mef2c-Cre and Prdm1-Cre mice to study OFT morphogenesis and septation, making a comparison between these two Cre lines. New insights were obtained in understanding SHF development including differentiation into cardiomyocytes in the OFT using Prdm1-Cre mice. In conclusion, we found that Prdm1-Cre mouse line is a more appropriate tool to monitor SHF development, while the Mef2c-Cre mice are excellent in studying the role and function of the SHF in OFT morphogenesis and septation.
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Corazón , Integrasas , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Animales , Ratones , Corazón/embriología , Integrasas/metabolismo , Integrasas/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones Transgénicos , Regulación del Desarrollo de la Expresión Génica/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Técnicas de Sustitución del GenRESUMEN
Cilia of higher animals sense various environmental stimuli. Proper ciliary signaling requires appropriate extent of BBSome-mediated export of membrane receptors across ciliary barrier transition zone (TZ) through retrograde intraflagellar transport (IFT) machinery. How the barrier passage is controlled, however, remains unknown. Here, we show that small GTPase Rabl2 functions as a molecular switch for the outward TZ passage. Rabl2-GTP enters cilia by binding to IFT-B complex. Its GTP hydrolysis enables the outward TZ passage of the BBSome and its cargos with retrograde IFT machinery, whereas its persistent association leads to their shedding from IFT-B during the passing process and consequently ciliary retention. Rabl2 deficiency or expression of a GTP-locked mutant impairs the ciliary hedgehog signaling without interfering with ciliation and respectively results in different spectrums of mouse developmental disorders. We propose that the switch role of Rabl2 ensures proper turnover of the BBSome and ciliary membrane receptors to fine-tune cilia-dependent signaling for normal embryonic development and organismic homeostasis.
Asunto(s)
Cilios/metabolismo , Guanosina Trifosfato/metabolismo , Transporte de Proteínas/fisiología , Transducción de Señal/fisiología , Proteínas de Unión al GTP rab/metabolismo , Animales , Línea Celular , Desarrollo Embrionario/fisiología , Flagelos/metabolismo , Células HEK293 , Proteínas Hedgehog/metabolismo , Homeostasis/fisiología , Humanos , Hidrólisis , Ratones , Unión Proteica/fisiologíaRESUMEN
Current understanding holds that Klinefelter syndrome (KS) is not inherited, but arises randomly during meiosis. Whether there is any genetic basis for the origin of KS is unknown. Here, guided by our identification of some USP26 variations apparently associated with KS, we found that knockout of Usp26 in male mice resulted in the production of 41, XXY offspring. USP26 protein is localized at the XY body, and the disruption of Usp26 causes incomplete sex chromosome pairing by destabilizing TEX11. The unpaired sex chromosomes then result in XY aneuploid spermatozoa. Consistent with our mouse results, a clinical study shows that some USP26 variations increase the proportion of XY aneuploid spermatozoa in fertile men, and we identified two families with KS offspring wherein the father of the KS patient harbored a USP26-mutated haplotype, further supporting that paternal USP26 mutation can cause KS offspring production. Thus, some KS should originate from XY spermatozoa, and paternal USP26 mutations increase the risk of producing KS offspring.
Asunto(s)
Cisteína Endopeptidasas/genética , Síndrome de Klinefelter/genética , Mutación/genética , Adulto , Aneuploidia , Animales , Humanos , Masculino , Ratones , Ratones Noqueados , Cromosomas Sexuales/genética , Espermatozoides/patología , Adulto JovenRESUMEN
BACKGROUND: Response to immunotherapy is the main challenge of head and neck squamous cancer (HNSCC) treatment. Previous studies have indicated that tumor mutational burden (TMB) is associated with prognosis, but it is not always a precise index. Hence, investigating specific genetic mutations and tumor microenvironment (TME) changes in TMB-high patients is essential for precision therapy of HNSCC. METHODS: A total of 33 HNSCC patients were enrolled in this study. We calculated the TMB score based on next-generation sequencing (NGS) sequencing and grouped these patients based on TMB score. Then, we examined the immune microenvironment of HNSCC using assessments of the bulk transcriptome and the single-cell RNA sequence (scRNA-seq) focusing on the molecular nature of TMB and mutations in HNSCC from our cohort. The association of the mutation pattern and TMB was analyzed in The Cancer Genome Atlas (TCGA) and validated by our cohort. RESULTS: 33 HNSCC patients were divided into three groups (TMB-low, -medium, and -high) based on TMB score. In the result of 520-gene panel sequencing data, we found that FAT1 and LRP1B mutations were highly prevalent in TMB-high patients. FAT1 mutations are associated with resistance to immunotherapy in HNSCC patients. This involves many metabolism-related pathways like RERE, AIRE, HOMER1, etc. In the scRNA-seq data, regulatory T cells (Tregs), monocytes, and DCs were found mainly enriched in TMB-high samples. CONCLUSION: Our analysis unraveled the FAT1 gene as an assistant predictor when we use TMB as a biomarker of drug resistance in HNSCC. Tregs, monocytes, and dendritic cells (DCs) were found mainly enriched in TMB-high samples.
Asunto(s)
Neoplasias de Cabeza y Cuello , Inmunoterapia , Mutación , Carcinoma de Células Escamosas de Cabeza y Cuello , Microambiente Tumoral , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Microambiente Tumoral/inmunología , Microambiente Tumoral/genética , Inmunoterapia/métodos , Masculino , Femenino , Persona de Mediana Edad , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/terapia , Neoplasias de Cabeza y Cuello/patología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Anciano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Pronóstico , Proteínas de la Membrana/genética , CadherinasRESUMEN
Although MRPS16 is involved in cancer development, its mechanisms in developing LAUD remain unclear. Herein, qRT-PCR, WB and IHC were utilized for evaluating MRPS16 expression levels, while functional assays besides animal experiments were performed to measure MRPS16 effect on LAUD progression. Using WB, the MRPS16 effect on PI3K/AKT/Frataxin signalling pathway was tested. According to our study, MRPS16 was upregulated in LAUD and was correlated to the advanced TNM stage as well as poor clinical outcomes, which represent an independent prognostic factor. Based on functional assays, MRPS16 is involved in promoting LAUD growth, migration and invasion, which was validated further in subsequent analyses through PI3K/AKT/Frataxin pathway activation. Moreover, MRPS16-knockdown-mediated Frataxin overexpression was shown to restore the reduction in tumour cells proliferation, migration and invasion. Our results revealed that MRPS16 caused an aggressive phenotype to LAUD and was a poor prognosticator; thus, targeting MRPS16 may be effectual in LAUD treatment.
Asunto(s)
Adenocarcinoma del Pulmón , Adenocarcinoma , Neoplasias Pulmonares , Animales , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Frataxina , Línea Celular Tumoral , Proliferación Celular/genética , Adenocarcinoma del Pulmón/genética , Neoplasias Pulmonares/patología , Movimiento Celular/genéticaRESUMEN
Adenine base editors (ABEs) are genome-editing tools that have been harnessed to introduce precise Aâ¢T to Gâ¢C conversion. The discovery of split genes revealed that all introns contain two highly conserved dinucleotides, canonical "AG" (acceptor) and "GT" (donor) splice sites. ABE can directly edit splice acceptor sites of the adenine (A) base, leading to aberrant gene splicing, which may be further adopted to remodel splicing. However, spliced isoforms triggered with ABE have not been well explored. To address it, we initially generated a cell line harboring C-terminal enhanced GFP (eGFP)-tagged ß-actin (ACTB), in which the eGFP signal can track endogenous ß-actin expression. Expectedly, after the editing of splice acceptor sites, we observed a dramatical decrease in the percentage of eGFP-positive cells and generation of splicing products with the noncanonical splice site. Furthermore, we manipulated Peroxidasin in mouse embryos with ABE, in which a noncanonical acceptor was activated to remodel splicing, successfully generating a mouse disease model of anophthalmia and severely malformed microphthalmia. Collectively, we demonstrate that ABE-mediated splicing remodeling can activate a noncanonical acceptor to manipulate human and mouse genomes, which will facilitate the investigation of basic and translational medicine studies.
Asunto(s)
Adenina , Sitios de Empalme de ARN , Animales , Humanos , Ratones , Actinas/genética , Secuencia de Bases , Edición Génica , Intrones , Empalme del ARN , Células HEK293RESUMEN
Lgr5+ intestinal stem cells (ISCs) exhibit self-renewal and differentiation features under homeostatic conditions, but the mechanisms controlling Lgr5 + ISC self-renewal remain elusive. Here, we show that the chromatin remodeler SRCAP is highly expressed in mouse intestinal epithelium and ISCs. Srcap deletion impairs both self-renewal of ISCs and intestinal epithelial regeneration. Mechanistically, SRCAP recruits the transcriptional regulator REST to the Prdm16 promoter and induces expression of this transcription factor. By activating PPARδ expression, Prdm16 in turn initiates PPARδ signaling, which sustains ISC stemness. Rest or Prdm16 deficiency abrogates the self-renewal capacity of ISCs as well as intestinal epithelial regeneration. Collectively, these data show that the SRCAP-REST-Prdm16-PPARδ axis is required for self-renewal maintenance of Lgr5 + ISCs.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Mucosa Intestinal/enzimología , Transducción de Señal , Células Madre/enzimología , Adenosina Trifosfatasas/genética , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Humanos , Mucosa Intestinal/citología , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments.
Asunto(s)
Astenozoospermia/genética , Infertilidad Masculina/genética , Animales , Astenozoospermia/patología , Astenozoospermia/fisiopatología , Estudios de Cohortes , Femenino , Eliminación de Gen , Genes Ligados a X , Hemicigoto , Humanos , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Infertilidad Masculina/fisiopatología , Masculino , Ratones Endogámicos C57BL , Mutación , Mutación Missense , Linaje , Fenotipo , Inyecciones de Esperma Intracitoplasmáticas , Motilidad Espermática , Cola del Espermatozoide/ultraestructura , Espermatozoides/patología , Espermatozoides/fisiología , Espermatozoides/ultraestructura , Secuenciación del ExomaRESUMEN
Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood owing to the lack of adequate methods for distinguishing circRNAs from cognate messenger RNAs with overlapping exons. Here, we report that CRISPR-RfxCas13d can effectively discriminate circRNAs from mRNAs by using guide RNAs targeting sequences spanning back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type-specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation by preventing the mRNA for family with sequence similarity 120A (FAM120A) from binding the translation inhibitor IGF2BP2. Further application of RfxCas13d-BSJ-gRNA screening has uncovered circMan1a2, which has regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR-RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.