RESUMEN

Male infertility is a complex multifactorial reproductive disorder with highly heterogeneous phenotypic presentations. Azoospermia is a medically non-manageable cause of male infertility affecting ∼1% of men. Precise etiology of azoospermia is not known in approximately three-fourth of the cases. To explore the genetic basis of azoospermia, we performed whole exome sequencing in two non-obstructive azoospermia affected siblings from a consanguineous Pakistani family. Bioinformatic filtering and segregation analysis of whole exome sequencing data resulted in the identification of a rare homozygous missense variant (c.962G>C, p. Arg321Thr) in YTHDC2, segregating with disease in the family. Structural analysis of the missense variant identified in our study and two previously reported functionally characterized missense changes (p. Glu332Gln and p. His327Arg) in mice showed that all these three variants may affect Mg2+ binding ability and helicase activity of YTHDC2. Collectively, our genetic analyses and experimental observations revealed that missense variant of YTHDC2 can induce azoospermia in humans. These findings indicate the important role of YTHDC2 deficiency for azoospermia and will provide important guidance for genetic counseling of male infertility.

Asunto(s)

Azoospermia , Secuenciación del Exoma , Homocigoto , Mutación Missense , Linaje , Hermanos , Adulto , Animales , Humanos , Masculino , Ratones , Azoospermia/genética , Azoospermia/patología , Consanguinidad , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Pakistán , ARN Helicasas/genética

RESUMEN

Aberrant long non-coding RNA (lncRNA) expression has been shown to be involved in the pathological process of pre-eclampsia (PE), yet only a small portion of lncRNAs has been characterized concerning the function and molecular mechanisms involved in PE. This study aimed to investigate the regulatory mechanism of the lncRNA AC092100.1 (AC092100.1) in angiogenesis in PE. In our study, bioinformatics analysis was performed to screen for differentially expressed lncRNAs between normal subjects and PE patients. The levels of AC092100.1 in placental tissues of patients with or without PE were validated using qRT-PCR. The effect of AC092100.1 overexpression on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) was investigated. The binding of AC092100.1 and YT521-B homology domain-containing 2 (YTHDC2) was predicted and verified. The effect of AC092100.1/YTHDC2 on the expression of vascular endothelial growth factor-A (VEGFA) in HUVECs was determined. Finally, a PE mice model was conducted. Fetal mouse growth, the abundance of mesenchymal morphology markers, including hypoxia-inducible factor 1-alpha (HIF-1α), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), Slug, and Vimentin, and endothelial markers, including placental growth factor (PLGF), CD31, and vascular endothelial (VE)-cadherin, in placental tissues were assessed. Here, we found that AC092100.1 was abnormally downregulated in placental tissues from PE patients. We established that AC092100.1 overexpression promoted HUVEC proliferation, migration, and tube formation in vitro. Mechanistically, AC092100.1 induced the accumulation of YTHDC2 and VEGFA through binding to YTHDC2 in HUVECs. Inhibition of YTHDC2 or VEGFA reversed AC092100.1-promoted tube formation. AC092100.1 overexpression contributed to alleviating fetal growth disorder, decreased levels of sEng, HIF-1α, sFlt-1, Slug, and Vimentin, and increased levels of VEGFA, PLGF, CD31, and VE-cadherin in PE mice. Our findings provided evidence supporting the role of the AC092100.1/YTHDC2/VEGFA axis in regulating angiogenesis, which demonstrated a therapeutic pathway for PE targeting angiogenesis.

Asunto(s)

Células Endoteliales de la Vena Umbilical Humana , Preeclampsia , ARN Largo no Codificante , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular , Preeclampsia/metabolismo , Preeclampsia/genética , Preeclampsia/patología , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , Femenino , Embarazo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ratones , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Proliferación Celular , Movimiento Celular , Neovascularización Patológica/metabolismo , Neovascularización Patológica/genética , Placenta/metabolismo , Angiogénesis

RESUMEN

BACKGROUND: A newly identified type of cell death due to intracellular copper accumulation is known as cuproptosis and RNA methylation is a post-transcriptional modification mechanism, both of which perform vital roles in the immune microenvironment of colorectal cancer (CRC), but the link between the two needs more research. METHODS: TCGA database provided RNA-seq data and details clinically of CRC samples. Cuproptosis-related RNA methylation regulators (CRRMRs) were identified by correlation analysis. We screened 6 CRRMRs for prognostic model construction by employing LASSO-Cox regression analysis and calculated risk scores by CRRMRs (CuMS). GSE39582 and GSE38832 cohort were used as external validation sets. This research concentrated on the connection between the prognostic model and somatic mutation, anti-cancer drug sensitivity, immune infiltration, immune checkpoint expression. In addition, we investigated the differential expression of YTHDC2 in epithelial cell subpopulations by single-cell analysis with GSE166555, calculated cuproptosis scores and performed pathway enrichment. In vitro experiments were performed to explore the consequences of knockdown of YTHDC2 on CRC cell proliferation and migration, as well as changes in CRC cell viability in response to elesclomol after knockdown of YTHDC2. In vivo experiments, we constructed the cell line-derived xenograft model to further validate the results of the in vitro experiments. RESULTS: The prognosis of CRC can be predicted by CuMS, which GSE39582 and GSE38832 confirmed. Two CuMS groups showed different tumor mutation burden (TMB) and immune infiltration. CuMS was connected to emerging immune checkpoints CD47 and PVR, therefore, it can be clinically complementary to TMB and microsatellite instability (MSI) status. In single-cell analysis, a subpopulation of epithelial cells with high YTHDC2 expression had a high cuproptosis score. In vitro experiments, knocking down YTHDC2 promoted cell proliferation and migration in CRC, and weaken the inhibitory effect of elesclomol and elesclomol-Cu on cell viability, which in vivo experiments validated. CONCLUSION: We developed a prognostic model constructed by 6 CRRMRs to assess overall survival and immune microenvironment of CRC patients. YTHDC2 might regulate cuproptosis in multiple ways.

Asunto(s)

Neoplasias Colorrectales , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Animales , Ratones , Regulación hacia Abajo , Proliferación Celular , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Microambiente Tumoral/inmunología , Pronóstico , Metilación , Movimiento Celular/genética , Ratones Desnudos , Femenino , Masculino , Metilación de ARN , ARN Helicasas

RESUMEN

Exposure to fine particulate matter (PM2.5) is a significant risk factor for hepatic steatosis. The N6-methyladenosine (m6A) is implicated in metabolic disturbances triggered by exogenous environmental factors. However, the role of m6A in mediating PM2.5-induced hepatic steatosis remains unclear. Herein, male C57BL/6J mice were subjected to PM2.5 exposure throughout the entire heating season utilizing a real-ambient PM2.5 whole-body inhalation exposure system. Concurrently, HepG2 cell models exposed to PM2.5 were developed to delve the role of m6A methylation modification. Following PM2.5 exposure, significant hepatic lipid accumulation and elevated global m6A level were observed both in vitro and in vivo. The downregulation of YTHDC2, an m6A-binding protein, might contribute to this alteration. In vitro studies revealed that lipid-related genes CEPT1 and YWHAH might be targeted by m6A modification. YTHDC2 could bind to CDS region of them and increase their stability. Exposure to PM2.5 shortened mRNA lifespan and suppressed the expression of CEPT1 and YWHAH, which were reversed to baseline or higher level upon the enforced expression of YTHDC2. Consequently, our findings indicate that PM2.5 induces elevated m6A methylation modification of CEPT1 and YWHAH by downregulating YTHDC2, which in turn mediates the decrease in the mRNA stabilization and expression of these genes, ultimately resulting in hepatic steatosis.

Asunto(s)

Adenosina , Hígado Graso , Ratones Endogámicos C57BL , Material Particulado , Proteínas de Unión al ARN , Animales , Humanos , Masculino , Ratones , Adenosina/análogos & derivados , Contaminantes Atmosféricos/toxicidad , Hígado Graso/inducido químicamente , Hígado Graso/genética , Hígado Graso/metabolismo , Células Hep G2 , Hígado/metabolismo , Hígado/efectos de los fármacos , Material Particulado/toxicidad , ARN Helicasas , Metilación de ARN , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética

RESUMEN

The specialized cell cycle of meiosis transforms diploid germ cells into haploid gametes. In mammals, diploid spermatogenic cells acquire the competence to initiate meiosis in response to retinoic acid. Previous mouse studies revealed that MEIOC interacts with RNA-binding proteins YTHDC2 and RBM46 to repress mitotic genes and to promote robust meiotic gene expression in spermatogenic cells that have initiated meiosis. Here, we have used the enhanced resolution of scRNA-seq and bulk RNA-seq of developmentally synchronized spermatogenesis to define how MEIOC molecularly supports early meiosis in spermatogenic cells. We demonstrate that MEIOC mediates transcriptomic changes before meiotic initiation, earlier than previously appreciated. MEIOC, acting with YTHDC2 and RBM46, destabilizes its mRNA targets, including the transcriptional repressors E2f6 and Mga, in mitotic spermatogonia. MEIOC thereby derepresses E2F6- and MGA-repressed genes, including Meiosin and other meiosis-associated genes. This confers on spermatogenic cells the molecular competence to, in response to retinoic acid, fully activate the transcriptional regulator STRA8-MEIOSIN, which is required for the meiotic G1/S phase transition and for meiotic gene expression. We conclude that, in mice, mRNA decay mediated by MEIOC-YTHDC2-RBM46 enhances the competence of spermatogenic cells to initiate meiosis.

Asunto(s)

Meiosis , ARN Mensajero , Proteínas de Unión al ARN , Espermatogénesis , Animales , Masculino , Ratones , ARN Mensajero/metabolismo , ARN Mensajero/genética , Espermatogénesis/genética , Espermatogénesis/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Espermatogonias/metabolismo , Espermatogonias/citología , Tretinoina/metabolismo , Tretinoina/farmacología , Estabilidad del ARN/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , ARN Helicasas

RESUMEN

N6-methyladenosine (m6A) stands as the most prevalent modified form of RNA in eukaryotes, pivotal in various biological processes such as regulating RNA stability, translation, and transcription. All members within the YT521-B homology (YTH) gene family are categorized as m6A reading proteins, capable of identifying and binding m6A modifications on RNA, thereby regulating RNA metabolism and functioning across diverse physiological processes. YTH domain-containing 2 (YTHDC2), identified as the latest member of the YTH family, has only recently started to emerge for its biological function. Numerous studies have underscored the significance of YTHDC2 in human physiology, highlighting its involvement in both tumor progression and non-tumor diseases. Consequently, this review aims to further elucidate the pathological mechanisms of YTHDC2 by summarizing its functions and roles in tumors and other diseases, with a particular focus on its downstream molecular targets and signaling pathways.

Asunto(s)

Adenosina , Neoplasias , Proteínas de Unión al ARN , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteínas de Unión al ARN/metabolismo , Animales , Enfermedad , Transducción de Señal , ARN Helicasas

RESUMEN

OBJECTIVE: To find the relationship between N6-methyladenosine (m6A) genes and Major Depressive Disorder (MDD). METHODS: Differential expression of m6A associated genes between normal and MDD samples was initially identified. Subsequent analysis was conducted on the functions of these genes and the pathways they may affect. A diagnostic model was constructed using the expression matrix of these differential genes, and visualized using a nomogram. Simultaneously, an unsupervised classification method was employed to classify all patients based on the expression of these m6A associated genes. Following this, common differential genes among different clusters were computed. By analyzing the functions of the common differential expressed genes among clusters, the role of m6A-related genes in the pathogenesis of MDD patients was elucidated. RESULTS: Differential expression was observed in ELAVL1 and YTHDC2 between the MDD group and the control group. ELAVL1 was associated with comorbid anxiety in MDD patients. A linear regression model based on these two genes could accurately predict whether patients in the GSE98793 dataset had MDD and could provide a net benefit for clinical decision-making. Based on the expression matrix of ELAVL1 and YTHDC2, MDD patients were classified into three clusters. Among these clusters, there were 937 common differential genes. Enrichment analysis was also performed on these genes. The ssGSEA method was applied to predict the content of 23 immune cells in the GSE98793 dataset samples. The relationship between these immune cells and ELAVL1, YTHDC2, and different clusters was analyzed. CONCLUSION: Among all the m6A genes, ELAVL1 and YTHDC2 are closely associated with MDD, ELAVL1 is related to comorbid anxiety in MDD. ELAVL1 and YTHDC2 have opposite associations with immune cells in MDD.

Asunto(s)

Adenosina , Trastorno Depresivo Mayor , Humanos , Trastorno Depresivo Mayor/genética , Adenosina/análogos & derivados , Adenosina/genética , Femenino , Masculino , Metilación , Proteínas de Unión al ARN/genética , Adulto , Nomogramas , ARN Helicasas

RESUMEN

BACKGROUND: Liver ischemia/reperfusion (I/R) injury is usually caused by hepatic inflow occlusion during liver surgery, and is frequently observed during war wounds and trauma. Hepatocyte ferroptosis plays a critical role in liver I/R injury, however, it remains unclear whether this process is controlled or regulated by members of the DEAD/DExH-box helicase (DDX/DHX) family. METHODS: The expression of DDX/DHX family members during liver I/R injury was screened using transcriptome analysis. Hepatocyte-specific Dhx58 knockout mice were constructed, and a partial liver I/R operation was performed. Single-cell RNA sequencing (scRNA-seq) in the liver post I/R suggested enhanced ferroptosis by Dhx58hep-/-. The mRNAs and proteins associated with DExH-box helicase 58 (DHX58) were screened using RNA immunoprecipitation-sequencing (RIP-seq) and IP-mass spectrometry (IP-MS). RESULTS: Excessive production of reactive oxygen species (ROS) decreased the expression of the IFN-stimulated gene Dhx58 in hepatocytes and promoted hepatic ferroptosis, while treatment using IFN-α increased DHX58 expression and prevented ferroptosis during liver I/R injury. Mechanistically, DHX58 with RNA-binding activity constitutively associates with the mRNA of glutathione peroxidase 4 (GPX4), a central ferroptosis suppressor, and recruits the m6A reader YT521-B homology domain containing 2 (YTHDC2) to promote the translation of Gpx4 mRNA in an m6A-dependent manner, thus enhancing GPX4 protein levels and preventing hepatic ferroptosis. CONCLUSIONS: This study provides mechanistic evidence that IFN-α stimulates DHX58 to promote the translation of m6A-modified Gpx4 mRNA, suggesting the potential clinical application of IFN-α in the prevention of hepatic ferroptosis during liver I/R injury.

Asunto(s)

Ferroptosis , Daño por Reperfusión , Animales , Ratones , Diclorodifenil Dicloroetileno , Hepatocitos , Interferón-alfa , ARN , ARN Mensajero

RESUMEN

Oxidative stress and neuronal dysfunction caused by intracerebral haemorrhage (ICH) can lead to secondary injury. The m6A modification has been implicated in the progression of ICH. This study aimed to investigate the role of the m6A reader YTHDC2 in ICH-induced secondary injury. ICH models were established in rats using autologous blood injection, and neuronal cell models were induced with Hemin. Experiments were conducted to overexpress YTH domain containing 2 (YTHDC2) and examine its effects on neuronal dysfunction, brain injury, and neuronal ferritinophagy. RIP-qPCR and METTL3 silencing were performed to investigate the regulation of YTHDC2 on nuclear receptor coactivator 4 (NCOA4). Finally, NCOA4 overexpression was used to validate the regulatory mechanism of YTHDC2 in ICH. The study found that YTHDC2 expression was significantly downregulated in the brain tissues of ICH rats. However, YTHDC2 overexpression improved neuronal dysfunction and reduced brain water content and neuronal death after ICH. Additionally, it reduced levels of ROS, NCOA4, PTGS2, and ATG5 in the brain tissues of ICH rats, while increasing levels of FTH and FTL. YTHDC2 overexpression also decreased levels of MDA and Fe2+ in the serum, while promoting GSH synthesis. In neuronal cells, YTHDC2 overexpression alleviated Hemin-induced injury, which was reversed by Erastin. Mechanistically, YTHDC2-mediated m6A modification destabilized NCOA4 mRNA, thereby reducing ferritinophagy and alleviating secondary injury after ICH. However, the effects of YTHDC2 were counteracted by NCOA4 overexpression. Overall, YTHDC2 plays a protective role in ICH-induced secondary injury by regulating NCOA4-mediated ferritinophagy.

Asunto(s)

Adenina , Lesiones Encefálicas , Hemina , Animales , Ratas , Adenina/análogos & derivados , Lesiones Encefálicas/genética , Lesiones Encefálicas/metabolismo , Hemorragia Cerebral/genética , Hemorragia Cerebral/metabolismo , Metilación de ADN , Hemina/farmacología , Hemina/metabolismo , Estrés Oxidativo , Factores de Transcripción/metabolismo

RESUMEN

Talaromyces marneffei (TM) immune evasion is an important factor leading to the high mortality rate of Penicilliosis marneffei. N6 -methyladenosine (m6 A) plays important roles in host immune response to various pathogen infections, yet its role in TM and HIV/TM coinfection remains largely unexplored. Here we reported genome-wide transcriptional m6 A profiles of TM mono-infection and HIV/TM coinfection. Our finding revealed dynamic alterations in global m6 A levels and upregulation of the m6 A reader YTH N6 -methyladenosine RNA binding protein C2 (YTHDC2) in TM-infected macrophages. Knockdown of YTHDC2 in TM-infected cells showed an elevated expression of TLR2 through m6 A-dependence, along with upregulation of TNF-α and IL1-ß. Overall, we characterized the m6 A profiles of the host and fungus before and after TM infection, and demonstrated that YTHDC2 mediates the key m6 A site of TLR2 to exert its function. These findings provide new insights into the underlying mechanisms and novel therapeutic approaches for TM diseases.

Asunto(s)

Coinfección , Infecciones por VIH , Micosis , Humanos , Receptor Toll-Like 2/genética , ARN Helicasas

RESUMEN

Ultraviolet B (UVB) radiation represents a major carcinogen for the development of all skin cancer types. Mechanistically, UVB induces damage to DNA in the form of lesions, including cyclobutane pyrimidine dimers (CPDs). Disruption of the functional repair processes, such as nucleotide excision repair (NER), allows persistence of DNA damage and contributes to skin carcinogenesis. Recent work has implicated m6A RNA methylation and its regulatory proteins as having critical roles in facilitating UVB-induced DNA damage repair. However, the biological functions of the m6A reader YTHDC2 are unknown in this context. Here, we show that YTHDC2 inhibition enhances the repair of UVB-induced DNA damage. We discovered that YTHDC2 inhibition increased the expression of PTEN while it decreased the expression of the PRC2 component SUZ12 and the levels of the histone modification H3K27me3. However, none of these functions were causally linked to the improvements in DNA repair, suggesting that the mechanism utilized by YTHDC2 may be unconventional. Moreover, inhibition of the m6A writer METTL14 reversed the effect of YTHDC2 inhibition on DNA repair while inhibition of the m6A eraser FTO mimicked the effect of YTHDC2 inhibition, indicating that YTHDC2 may regulate DNA repair through the m6A pathway. Finally, compared to normal human skin, YTHDC2 expression was upregulated in human cutaneous squamous cell carcinomas (cSCC), suggesting that it may function as a tumor-promoting factor in skin cancer. Taken together, our findings demonstrate that the m6A reader YTHDC2 plays a role in regulating UVB-induced DNA damage repair and may serve as a potential biomarker in cSCC.

Asunto(s)

Daño del ADN , Reparación del ADN , Rayos Ultravioleta , Humanos , Histonas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , ARN Helicasas

RESUMEN

N6-Methyladenosine (m6A) mRNA methylation modification is regarded as an important mechanism involved in diverse physiological processes. YT521-B homology (YTH) domain family members are associated with the tumorigenesis of several cancers. However, the role of YTHDC2 in papillary thyroid cancer (PTC) progression remains unknown. Results showed that YTHDC1, YTHDF1, YTHDF2, and YTHDF3 showed no observable difference in thyroid cancer samples. YTHDC2 was significantly downregulated in thyroid cancer samples and cells. YTHDC2 inhibited cell proliferation in PTC cells. YTHDC2 elicited apoptosis in PTC cells, as demonstrated by the elevated expression of pro-apoptotic factors cl-caspase-3/caspase-3 and Bcl-2-associated (Bax), and the reduced anti-apoptotic B cell lymphoma-2 (Bcl-2) expression. There was a positive correlation between YTHDC2 and cylindromatosis (CYLD) expression based on GEPIA database. YTHDC2 increased CYLD expression in PTC cells. CYLD knockdown abolished the effects of YTHDC2 on PTC cell proliferation and apoptosis. Additionally, YTHDC2 inactivated the protein kinase B (Akt) pathway by increasing CYLD in PTC cells. Overall, YTHDC2 inhibited cell proliferation and induced apoptosis in PTC cells by regulating CYLD-mediated inactivation of Akt pathway.

Asunto(s)

Proteínas Proto-Oncogénicas c-akt , Neoplasias de la Tiroides , Humanos , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Caspasa 3/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Proliferación Celular/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Apoptosis/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Enzima Desubiquitinante CYLD/genética , Enzima Desubiquitinante CYLD/metabolismo , ARN Helicasas

RESUMEN

Inherited retinal dystrophies (IRDs) are major causes of visual impairment and irreversible blindness worldwide, while the precise molecular and genetic mechanisms are still elusive. N6-methyladenosine (m6A) modification is the most prevalent internal modification in eukaryotic mRNA. YTH domain containing 2 (YTHDC2), an m6A reader protein, has recently been identified as a key player in germline development and human cancer. However, its contribution to retinal function remains unknown. Here, we explore the role of YTHDC2 in the visual function of retinal rod photoreceptors by generating rod-specific Ythdc2 knockout mice. Results show that Ythdc2 deficiency in rods causes diminished scotopic ERG responses and progressive retinal degeneration. Multi-omics analysis further identifies Ppef2 and Pde6b as the potential targets of YTHDC2 in the retina. Specifically, via its YTH domain, YTHDC2 recognizes and binds m6A-modified Ppef2 mRNA at the coding sequence and Pde6b mRNA at the 5'-UTR, resulting in enhanced translation efficiency without affecting mRNA levels. Compromised translation efficiency of Ppef2 and Pde6b after YTHDC2 depletion ultimately leads to decreased protein levels in the retina, impaired retinal function, and progressive rod death. Collectively, our finding highlights the importance of YTHDC2 in visual function and photoreceptor survival, which provides an unreported elucidation of IRD pathogenesis via epitranscriptomics.

Asunto(s)

Células Fotorreceptoras de Vertebrados , Degeneración Retiniana , Animales , Humanos , Ratones , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , ARN Helicasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

N6-methyladenosine (m6A) is the most common posttranscriptional RNA modification and plays significant roles in physiological and pathological progression. Here, we probed the functions and mechanism of the m6A reader YTH domain containing 2 (YTHDC2) in Lung Adenocarcinoma (LUAD) tumorigenesis. Levels of genes and proteins of YTHDC2 and Mitochondrial ribosomal protein L7/L12 (MRPL12) were assayed by quantitative real-time polymerase chain reaction, western blotting and Immunohistochemistry (IHC) analyses. In vitro analysis was conducted using 5-ethynyl-2'-deoxyuridine (EdU), colony formation, flow cytometry, and transwell assays, respectively. In vivo assay was performed by using the mouse lung adenocarcinoma model. The methylated RNA immunoprecipitation (MeRIP) assay was used to detect the m6A modification profile of MRPL12 mRNA. YTHDC2 was lowly expressed in lung adenocarcinoma tissues and cells. Overexpression of YTHDC2 suppressed the proliferation, invasion and migration of lung adenocarcinoma cells, but induced cell apoptosis. As expected, forced expression of YTHDC2 hindered lung adenocarcinoma tumor growth in vivo. Mechanistically, YTHDC2 preferentially bound to m6A-modified MRPL12 mRNA and destabilized its expression. MRPL12 was highly expressed in lung adenocarcinoma tissues and cells, and MRPL12 silencing repressed the growth and mobility of lung adenocarcinoma cells. Moreover, MRPL12 upregulation attenuated the anticancer activity of YTHDC2 in lung adenocarcinoma cells. In vivo assay also showed YTHDC2 suppressed tumor growth in the lung adenocarcinoma mouse model via downregulating MRPL12. The m6A reader YTHDC2 repressed lung adenocarcinoma tumorigenesis by destabilizing MRPL12 in an m6A-dependent manner.

Asunto(s)

Adenocarcinoma del Pulmón , Adenosina , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares , Humanos , Animales , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Línea Celular Tumoral , Carcinogénesis/genética , Apoptosis , Movimiento Celular , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Helicasas/metabolismo , ARN Helicasas/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Ratones Desnudos

RESUMEN

The innate immune response must be terminated in a timely manner at the late stage of infection to prevent unwanted inflammation. The role of m6A-modified RNAs and their binding partners in this process is not well known. Here, we develop an enzymolysis-based RNA pull-down (eRP) method that utilizes the immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) to fish out m6A-modified RNA-associated proteins. We apply eRP to capture the methylated single-stranded RNA (ssRNA) probe-associated proteins and identify YT521-B homology domain-containing 2 (YTHDC2) as the m6A-modified interferon ß (IFN-ß) mRNA-binding protein. YTHDC2, induced in macrophages at the late stage of virus infection, recruits IFN-stimulated exonuclease ISG20 (IFN-stimulated exonuclease gene 20) to degrade IFN-ß mRNA, consequently inhibiting antiviral innate immune response. In vitro and in vivo deficiency of YTHDC2 increases IFN-ß production at the late stage of viral infection. Our findings establish an eRP method to effectively identify RNA-protein interactions and add mechanistic insight to the termination of innate response for maintaining homeostasis.

Asunto(s)

Exorribonucleasas , Virosis , Animales , Exorribonucleasas/metabolismo , ARN Viral/genética , Exonucleasas/genética , Exonucleasas/metabolismo , Inmunidad Innata , Antivirales/farmacología , ARN Mensajero

RESUMEN

Aberrant expression of adipogenic regulatory factors (ADIRF) in tumor cells is critical for tumor growth and metastasis. N6-methyladenosine (m6A) modifications have an important role in a variety of biological activities. Our study aimed to investigate the role of ADIRF in adenocarcinoma and to elucidate the regulatory role of m6A signaling on ADIRF. Differential expression of genes in tumor and normal tissues was analyzed using the LUAD dataset (GSE1987). The Kaplan-Meier method and receiver operating characteristic (ROC) curve analysis were performed to evaluate the prognostic and diagnostic value of ADIRF in LUAD. Loss-of-function or gain-of-function experiments were performed to study the effect of ADIRF on LUAD growth in vitro. The molecular mechanism of action of ADIRF in LUAD was confirmed using a dual-luciferase reporter system and MeRIP-qPCR. We identified a loss of ADIRF expression in LUAD tissues and cells. Furthermore, the restoration of ADIRF levels attenuated LUAD cell growth and metastasis in vitro. Mechanistically, an m6A "eraser," α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5), eliminated the ADIRF m6A modification motif and further blocked the binding of the YTH domain-containing 2 (YTHDC2)-binding protein to ADIRF. At the molecular level, ALKBH5 enrichment increased ADIRF mRNA levels and prevented the attenuation of ADIRF mRNA by YTHDC2. The effects of ALKBH5 overexpression could also extend to the inhibition of LUAD cell proliferation and metastasis. This study linked ADIRF with the m6A modifying regulators ALKBH5 and YTHDC2, providing a promising molecular intervention for LUAD and deepening the understanding of LUAD mechanisms.

Asunto(s)

Adenocarcinoma del Pulmón , Adenocarcinoma , Neoplasias Pulmonares , Humanos , Pronóstico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Adenosina , Biomarcadores

RESUMEN

As the most abundant internal mRNA modification, N6-methyladenosine (m6A) RNA methylation has been found to influence many biological events including bone mesenchymal stem cells (BMSCs) osteogenic differentiation. YTH N6-methyladenosine RNA binding protein C2 （YTHDC2） is an m6A reading protein with the ability to mediate the decay of combined methylated mRNA, however its role in BMSCs osteogenic differentiation remains unknown. In this study, we first found an increase of RUNX family transcription factor 2 （RUNX2） expression and a decrease of YTHDC2 expression during the process of BMSCs osteogenic differentiation. Furthermore, we transfected BMSCs with YTHDC2 interference fragment, resulting in an increased content of RUNX2 mRNA and protein inside BMSCs. Finally, through RNA Immunoprecipitation experiments, we confirmed that YTHDC2 protein can bind to RUNX2 mRNA and accelerate its decomposition. Moreover, the immunofluorescence staining also showed a negative correlation between YTHDC2 and RUNX2. In conclusion, during BMSCs osteogenic differentiation, YTHDC2 protein showed decreased expression, resulting in a higher level of RUNX2 (mRNA and protein) expression inside cells, indicating YTHDC2 as a promising molecular target for the regulation of BMSCs osteogenic differentiation.

RESUMEN

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. Recently, NLRP3 has been demonstrated to be closely related to RA. The objective of our research was to analyze the specific mechanism of NLRP3 in RA. The m6A levels of NLRP3 was detected with methylated RNA immunoprecipitation (MeRIP) kit. The mRNA and protein levels of related genes were tested with RT-qPCR and Western blot. The inflammatory factors levels were detected with ELISA kits. The cell proliferative ability was measured with CCK-8 and EdU staining assays. NLRP3 levels was prominently in synovial tissues and fibroblast-like synoviocytes (FLS) from RA patients. NLRP3 silencing suppressed FLS proliferation and inflammatory factor levels. Additionally, ALKBH5 was found to bind with NLRP3, and ALKBH5 silencing suppressed FLS proliferation and inflammatory factor levels while NLRP3 overexpressing neutralized the role of ALKBH5 in FLS. Furthermore, m6A modified induced by ALKBH5 suppressed NLRP3 mRNA level through YTHDC2 in RA, and NLRP3 is a hinge factor in RA progression.

Asunto(s)

Artritis Reumatoide , Sinoviocitos , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Artritis Reumatoide/metabolismo , Membrana Sinovial/metabolismo , Sinoviocitos/metabolismo , Factores de Transcripción/metabolismo , Proliferación Celular , Fibroblastos/metabolismo , Células Cultivadas , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Proteínas de Unión al ARN/metabolismo

RESUMEN

Background: The methylation of adenosines at the N6 position (N6-methyladenosine; m6A) is one of the most conserved internal RNA modifications. m6A can modulate the expression of oncogenes or tumor suppressor genes, as well as m6A levels and the expression and activity of m6A enzymes, thus influencing tumor progression and therapeutic response. This study investigates the role of YTHDC2-mediated m6A messenger RNA (mRNA) modification of Id3 in controlling cisplatin resistance in non-small cell lung cancer (NSCLC). Methods: The expression of the m6A reader protein YTHDC2 was detected in an NSCLC cisplatin-resistant cell line (A549/DDP) using real-time fluorescence quantitative polymerase chain reaction (qPCR). YTHDC2 overexpression plasmids were constructed and transfected into A549/DDP and A549 cells respectively. We performed qPCR and western blot (WB) to detect changes in YTHDC2 and Id3 expression, and the effects of YTHDC2 overexpression on proliferation, apoptosis, invasion, and migration of drug-resistant cells were assessed by cell counting kit-8 (CCK-8), flow cytometry, and transwell and scratch assays. The m6A modification of Id3 by YTHDC2 was clarified by m6A-immunoprecipitation-PCR (m6A-IP-PCR) assay. Results: The CLIPdb online database predicted that YTHDC2 might bind to Id3. The results of qPCR showed that YTHDC2 was downregulated in the NSCLC cisplatin-resistant cell line A549/DDP compared to the cisplatin-sensitive cell line A549. Overexpression of YTHDC2 increased the expression of Id3, and the methylation inhibitor 3-deazaadenosine abrogated the regulatory effect of YTHDC2 on Id3. YTHDC2 overexpression significantly inhibited A549/DDP cell proliferation, migration, and invasion, and promoted apoptosis by synergistically promoting the effects of Id3. m6A-IP-PCR analysis revealed that YTHDC2 could inhibit the m6A level of Id3 mRNA. Conclusions: To regulate the activity of Id3, YTHDC2 requires modifications to m6A, which ultimately inhibit cisplatin resistance in NSCLC.

RESUMEN

Methyltransferase like 3 (METTL3), a primary N6-methyladenosine (m6A) methyltransferase, has been implicated in various biological and pathological processes including immune responses. However, the functions and mechanisms of METTL3 in pathogenic T helper (Th)17 cells are poorly understood. Here we found significantly decreased METTL3 expression along with reduced m6A levels in eyeballs and T cells of experimental autoimmune uveitis (EAU). Overexpression of METTL3 ameliorated the development of EAU and suppressed pathogenic Th17 cell responses in vivo and in vitro. Mechanistically, METTL3 promoted the expression of absent, small, or homeotic-like 1 (ASH1L) via enhancing its stability in a YT521-B homology domain containing 2 (YTHDC2)-dependent manner, which further decreased the expression of IL-17 and IL-23 receptor (IL-23R), resulting in reduced pathogenic Th17 responses. Together, our data reveal a pivotal role of METTL3 in regulating pathogenic Th17 responses, which may contribute to human uveitis therapy.

Asunto(s)

Proteínas de Unión al ADN , N-Metiltransferasa de Histona-Lisina , Metiltransferasas , Células Th17 , Uveítis , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , ARN Mensajero/genética , Uveítis/genética , Uveítis/metabolismo , Animales , Enfermedades Autoinmunes , Modelos Animales de Enfermedad