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1.
Mol Cell ; 83(12): 1956-1958, 2023 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-37327770

RESUMO

In this issue of Molecular Cell, Yu et al.1 identify RBM33 as a previously unrecognized m6A (N-6-methyladenosine) RNA binding protein that plays a critical role in ALKBH5-mediated m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5.


Assuntos
Adenosina , Homólogo AlkB 5 da RNA Desmetilase , Metilação , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adenosina/genética , Adenosina/metabolismo
2.
Mol Cell ; 83(12): 2003-2019.e6, 2023 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-37257451

RESUMO

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Neoplasias de Cabeça e Pescoço , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Carcinogênese
3.
Mol Cell ; 83(23): 4334-4351.e7, 2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-37979586

RESUMO

Growth factor receptors rank among the most important oncogenic pathways, but pharmacologic inhibitors often demonstrate limited benefit as monotherapy. Here, we show that epidermal growth factor receptor (EGFR) signaling repressed N6-methyladenosine (m6A) levels in glioblastoma stem cells (GSCs), whereas genetic or pharmacologic EGFR targeting elevated m6A levels. Activated EGFR induced non-receptor tyrosine kinase SRC to phosphorylate the m6A demethylase, AlkB homolog 5 (ALKBH5), thereby inhibiting chromosomal maintenance 1 (CRM1)-mediated nuclear export of ALKBH5 to permit sustained mRNA m6A demethylation in the nucleus. ALKBH5 critically regulated ferroptosis through m6A modulation and YTH N6-methyladenosine RNA binding protein (YTHDF2)-mediated decay of the glutamate-cysteine ligase modifier subunit (GCLM). Pharmacologic targeting of ALKBH5 augmented the anti-tumor efficacy of EGFR and GCLM inhibitors, supporting an EGFR-ALKBH5-GCLM oncogenic axis. Collectively, EGFR reprograms the epitranscriptomic landscape through nuclear retention of the ALKBH5 demethylase to protect against ferroptosis, offering therapeutic paradigms for the treatment of lethal cancers.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Receptores ErbB , Ferroptose , Glioblastoma , Humanos , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Receptores ErbB/genética , Ferroptose/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , RNA Mensageiro/genética
4.
Nat Immunol ; 18(10): 1094-1103, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28846086

RESUMO

DEAD-box (DDX) helicases are vital for the recognition of RNA and metabolism and are critical for the initiation of antiviral innate immunity. Modification of RNA is involved in many biological processes; however, its role in antiviral innate immunity has remained unclear. Here we found that nuclear DDX member DDX46 inhibited the production of type I interferons after viral infection. DDX46 bound Mavs, Traf3 and Traf6 transcripts (which encode signaling molecules involved in antiviral responses) via their conserved CCGGUU element. After viral infection, DDX46 recruited ALKBH5, an 'eraser' of the RNA modification N6-methyladenosine (m6A), via DDX46's DEAD helicase domain to demethylate those m6A-modified antiviral transcripts. It consequently enforced their retention in the nucleus and therefore prevented their translation and inhibited interferon production. DDX46 also suppressed antiviral innate immunity in vivo. Thus, DDX46 inhibits antiviral innate responses by entrapping selected antiviral transcripts in the nucleus by erasing their m6A modification, a modification normally required for export from the nucleus and translation.


Assuntos
Adenina/análogos & derivados , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Imunidade Inata/genética , Transcrição Gênica , Adenina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Interferon Tipo I/biossíntese , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Motivos de Nucleotídeos , Ligação Proteica , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estomatite Vesicular/genética , Estomatite Vesicular/imunologia , Estomatite Vesicular/metabolismo , Vesiculovirus/fisiologia , Replicação Viral
5.
Proc Natl Acad Sci U S A ; 121(6): e2312861121, 2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38285939

RESUMO

The N6-methyladenosine (m6A) modification of RNA is an emerging epigenetic regulatory mechanism that has been shown to participate in various pathophysiological processes. However, its involvement in modulating neuropathic pain is still poorly understood. In this study, we elucidate a functional role of the m6A demethylase alkylation repair homolog 5 (ALKBH5) in modulating trigeminal-mediated neuropathic pain. Peripheral nerve injury selectively upregulated the expression level of ALKBH5 in the injured trigeminal ganglion (TG) of rats. Blocking this upregulation in injured TGs alleviated trigeminal neuropathic pain, while mimicking the upregulation of ALKBH5 in intact TG neurons sufficiently induced pain-related behaviors. Mechanistically, histone deacetylase 11 downregulation induced by nerve injury increases histone H3 lysine 27 acetylation (H3K27ac), facilitating the binding of the transcription factor forkhead box protein D3 (FOXD3) to the Alkbh5 promoter and promoting Alkbh5 transcription. The increased ALKBH5 erases m6A sites in Htr3a messenger RNA (mRNA), resulting in an inability of YT521-B homology domain 2 (YTHDF2) to bind to Htr3a mRNA, thus causing an increase in 5-HT3A protein expression and 5-HT3 channel currents. Conversely, blocking the increased expression of ALKBH5 in the injured TG destabilizes nerve injury-induced 5-HT3A upregulation and reverses mechanical allodynia, and the effect can be blocked by 5-HT3A knockdown. Together, FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain through m6A-dependent stabilization of Htr3a mRNA in TG neurons. This mechanistic understanding may advance the discovery of new therapeutic targets for neuropathic pain management.


Assuntos
Neuralgia , Neuralgia do Trigêmeo , Animais , Ratos , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Neuralgia/genética , Neuralgia/metabolismo , RNA Mensageiro/metabolismo , Células Receptoras Sensoriais/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Receptores 5-HT3 de Serotonina/genética
6.
PLoS Pathog ; 20(1): e1011917, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38227578

RESUMO

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter (BCP). Here we show that the hypoxic-dependent increase in BCP-derived transcripts is dependent on N6-methyladenosine (m6A) modifications in the 5' stem loop that regulate RNA half-life. Application of a probe-enriched long-read sequencing method to accurately map the HBV transcriptome showed an increased abundance of pre-genomic RNA under hypoxic conditions. Mapping the transcription start sites of BCP-RNAs identified a role for hypoxia to regulate pre-genomic RNA splicing that is dependent on m6A modification. Bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2-NTCP cell line showed increased ALKBH5 gene expression under hypoxic conditions and a concomitant reduction in m6A-modified HBV BCP-RNA and host RNAs. Silencing the demethylase reduced the level of BCP-RNAs and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.


Assuntos
Vírus da Hepatite B , Hepatite B , Animais , Humanos , Camundongos , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Fucosiltransferases/genética , Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hipóxia , Oxigênio , RNA , Transcriptoma
7.
Genes Dev ; 32(23-24): 1472-1484, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30463905

RESUMO

Modification of mRNA by N6-adenosine methylation (m6A) on internal bases influences gene expression in eukaryotes. How the dynamic genome-wide landscape of m6A-modified mRNAs impacts virus infection and host immune responses remains poorly understood. Here, we show that type I interferon (IFN) production triggered by dsDNA or human cytomegalovirus (HCMV) is controlled by the cellular m6A methyltrasferase subunit METTL14 and ALKBH5 demethylase. While METTL14 depletion reduced virus reproduction and stimulated dsDNA- or HCMV-induced IFNB1 mRNA accumulation, ALKBH5 depletion had the opposite effect. Depleting METTL14 increased both nascent IFNB1 mRNA production and stability in response to dsDNA. In contrast, ALKBH5 depletion reduced nascent IFNB1 mRNA production without detectably influencing IFN1B mRNA decay. Genome-wide transcriptome profiling following ALKBH5 depletion identified differentially expressed genes regulating antiviral immune responses, while METTL14 depletion altered pathways impacting metabolic reprogramming, stress responses, and aging. Finally, we determined that IFNB1 mRNA was m6A-modified within both the coding sequence and the 3' untranslated region (UTR). This establishes that the host m6A modification machinery controls IFNß production triggered by HCMV or dsDNA. Moreover, it demonstrates that responses to nonmicrobial dsDNA in uninfected cells, which shape host immunity and contribute to autoimmune disease, are regulated by enzymes controlling m6A epitranscriptomic changes.


Assuntos
DNA/imunologia , Regulação da Expressão Gênica/genética , Sistema Imunitário/enzimologia , Imunidade Inata/genética , Interferon beta/genética , Metiltransferases/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Citomegalovirus/imunologia , Perfilação da Expressão Gênica , Humanos , Interferon beta/metabolismo , Estabilidade de RNA/genética , Células Vero , Replicação Viral/genética
8.
FASEB J ; 38(14): e23793, 2024 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-39003634

RESUMO

Sevoflurane, as a commonly used inhaled anesthetic for pediatric patients, has been reported that multiple sevoflurane exposures are associated with a greater risk of developing neurocognitive disorder. N6-Methyladenosine (m6A), as the most common mRNA modification in eukaryotes, has emerged as a crucial regulator of brain function in processes involving synaptic plasticity, learning and memory, and neurodevelopment. Nevertheless, the relevance of m6A RNA methylation in the multiple sevoflurane exposure-induced developmental neurotoxicity remains mostly elusive. Herein, we evaluated the genome-wide m6A RNA modification and gene expression in hippocampus of mice that received with multiple sevoflurane exposures using m6A-sequencing (m6A-seq) and RNA-sequencing (RNA-seq). We discovered 19 genes with differences in the m6A methylated modification and differential expression in the hippocampus. Among these genes, we determined that a total of nine differential expressed genes may be closely associated with the occurrence of developmental neurotoxicity induced by multiple sevoflurane exposures. We further found that the alkB homolog 5 (ALKBH5), but not methyltransferase-like 3 (METTL3) and Wilms tumor 1-associated protein (WTAP), were increased in the hippocampus of mice that received with multiple sevoflurane exposures. And the IOX1, as an inhibitor of ALKBH5, significantly improved the learning and memory defects and reduced neuronal damage in the hippocampus of mice induced by multiple sevoflurane exposures. The current study revealed the role of m6A methylated modification and m6A-related regulators in sevoflurane-induced cognitive impairment, which might provide a novel insight into identifying biomarkers and therapeutic strategies for inhaled anesthetic-induced developmental neurotoxicity.


Assuntos
Adenosina , Homólogo AlkB 5 da RNA Desmetilase , Hipocampo , Síndromes Neurotóxicas , Sevoflurano , Sevoflurano/toxicidade , Animais , Camundongos , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Masculino , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/prevenção & controle , Adenosina/análogos & derivados , Adenosina/metabolismo , Anestésicos Inalatórios/toxicidade , Camundongos Endogâmicos C57BL , Metilação/efeitos dos fármacos , Metiltransferases/metabolismo , Metiltransferases/genética
9.
Exp Cell Res ; 437(1): 113994, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38479704

RESUMO

m6A modification has been studied in tumors, but its role in host anti-tumor immune response and TAMs polarization remains unclear. The fatty acid oxidation (FAO) process of TAMs is also attracting attention. A co-culture model of colorectal cancer (CRC) cells and macrophages was used to simulate the tumor microenvironment. Expression changes of m6A demethylase genes FTO and ALKBH5 were screened. ALKBH5 was further investigated. Gain-of-function experiments were conducted to study ALKBH5's effects on macrophage M2 polarization, CRC cell viability, proliferation, migration, and more. Me-RIP and Actinomycin D assays were performed to study ALKBH5's influence on CPT1A, the FAO rate-limiting enzyme. AMP, ADP, and ATP content detection, OCR measurement, and ECAR measurement were used to explore ALKBH5's impact on macrophage FAO level. Rescue experiments validated ALKBH5's mechanistic role in macrophage M2 polarization and CRC malignant development. In co-culture, CRC cells enhance macrophage FAO and suppress m6A modification in M2 macrophages. ALKBH5 was selected as the gene for further investigation. ALKBH5 mediates CPT1A upregulation by removing m6A modification, promoting M2 macrophage polarization and facilitating CRC development. These findings indicate that ALKBH5 enhances fatty acid metabolism and M2 polarization of macrophages by upregulating CPT1A, thereby promoting CRC development.


Assuntos
Neoplasias Colorretais , Macrófagos , Humanos , Regulação para Cima/genética , Macrófagos/metabolismo , Neoplasias Colorretais/patologia , Ácidos Graxos/metabolismo , Microambiente Tumoral , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo
10.
Proc Natl Acad Sci U S A ; 119(33): e2203318119, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35939687

RESUMO

γδ T cells are an abundant T cell population at the mucosa and are important in providing immune surveillance as well as maintaining tissue homeostasis. However, despite γδ T cells' origin in the thymus, detailed mechanisms regulating γδ T cell development remain poorly understood. N6-methyladenosine (m6A) represents one of the most common posttranscriptional modifications of messenger RNA (mRNA) in mammalian cells, but whether it plays a role in γδ T cell biology is still unclear. Here, we show that depletion of the m6A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells, which confers enhanced protection against gastrointestinal Salmonella typhimurium infection. Mechanistically, loss of ALKBH5 favors the development of γδ T cell precursors by increasing the abundance of m6A RNA modification in thymocytes, which further reduces the expression of several target genes including Notch signaling components Jagged1 and Notch2. As a result, impairment of Jagged1/Notch2 signaling contributes to enhanced proliferation and differentiation of γδ T cell precursors, leading to an expanded mature γδ T cell repertoire. Taken together, our results indicate a checkpoint role of ALKBH5 and m6A modification in the regulation of γδ T cell early development.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Linfócitos Intraepiteliais , RNA Mensageiro , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Animais , Linfócitos Intraepiteliais/enzimologia , Linfócitos Intraepiteliais/imunologia , Proteína Jagged-1/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptor Notch2/metabolismo , Transdução de Sinais/genética
11.
J Cell Mol Med ; 28(2): e18066, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38098223

RESUMO

The long noncoding RNA PVT1 is reported to act as an oncogene in several kinds of cancers, especially ovarian cancer (OV). Abnormal levels of N6 -methyladenosine, a dynamic and reversible modification, are associated with tumorigenesis and malignancies. Our previous study reported that PVT1 plays critical roles in regulating OV. However, it is still largely unknown how m6 A modification affects OV via PVT1. In this study, we aimed to investigate the regulation of ALKBH5 by affecting PVT1 in OV. We first found that the PVT1 RNA level was higher in OV cells than in IOSE80 cells, and conversely, the m6 A modification level of PVT1 was lower in OV cells. By searching the HPA, ALKBH5, which is responsible for PVT1 demethylation, was found to be upregulated in OV tissues versus normal ovarian tissues. ALKBH5 binds to PVT1 RNA, and knockdown of ALKBH5 decreased PVT1 RNA levels. ALKBH5 also increased FOXM1 levels by upregulating PVT1, at least partially. Knockdown of ALKBH5 suppressed OV growth, colony formation, tumour formation and invasion, which were partially reversed by overexpression of PVT1. Moreover, ALKBH5 knockdown decreased FOXM1 levels by regulating PVT1 RNA expression, subsequently increasing the sensitivity to carboplatin, 5-FU and docetaxel chemotherapy. Taken together, these results indicate that ALKBH5 directly regulates the m6 A modification and stability of PVT1. Then, modified PVT1 further regulates FOXM1 and thus affects malignant behaviours and chemosensitivity in OV cells. All these results indicate that ALKBH5 regulates the malignant behaviour of OV by regulating PVT1/FOXM1.


Assuntos
Neoplasias Ovarianas , RNA Longo não Codificante , Humanos , Feminino , RNA Longo não Codificante/metabolismo , Proliferação de Células/genética , Neoplasias Ovarianas/patologia , Docetaxel , Carboplatina , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo
12.
J Biol Chem ; 299(8): 105071, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37474102

RESUMO

Paraspeckles (PS) are nuclear structures scaffolded by the long noncoding RNA NEAT1 and protein components such as NONO and SFPQ. We previously found that the upregulation of RNA N6-methyl-adenosine (m6A) demethylase ALKBH5 facilitates hypoxia-induced paraspeckle assembly through erasing m6A marks on NEAT1, thus stabilizing it. However, it remains unclear how these processes are spatiotemporally coordinated. Here we discover that ALKBH5 specifically binds to proteins in PS and forms phase-separated droplets that are incorporated into PS through its C-terminal intrinsically disordered region (cIDR). Upon exposure to hypoxia, rapid ALKBH5 condensation in PS induces m6A demethylation of NEAT1, which further facilitates PS formation before the upregulation of ALKBH5 expression. In cells expressing ALKBH5 lacking cIDR, PS fail to be formed in response to hypoxia, accompanied with insufficient m6A demethylation of NEAT1 and its destabilization. We also demonstrate that ALKBH5-cIDR is indispensable for hypoxia-induced effects such as cancer cell invasion. Therefore, our study has identified the role of ALKBH5 in phase separation as the molecular basis of the positive feedback loop for PS formation between ALKBH5 incorporation into PS and NEAT1 stabilization.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Paraspeckles , RNA Longo não Codificante , Humanos , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Hipóxia , Paraspeckles/metabolismo , RNA Longo não Codificante/genética , Ativação Transcricional , Regulação para Cima
13.
BMC Genomics ; 25(1): 634, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918701

RESUMO

BACKGROUND: Previous studies have demonstrated the role of N6-methyladenosine (m6A) RNA methylation in various biological processes, our research is the first to elucidate its specific impact on LCAT mRNA stability and adipogenesis in poultry. RESULTS: The 6 100-day-old female chickens were categorized into high (n = 3) and low-fat chickens (n = 3) based on their abdominal fat ratios, and their abdominal fat tissues were processed for MeRIP-seq and RNA-seq. An integrated analysis of MeRIP-seq and RNA-seq omics data revealed 16 differentially expressed genes associated with to differential m6A modifications. Among them, ELOVL fatty acid elongase 2 (ELOVL2), pyruvate dehydrogenase kinase 4 (PDK4), fatty acid binding protein 9 (PMP2), fatty acid binding protein 1 (FABP1), lysosomal associated membrane protein 3 (LAMP3), lecithin-cholesterol acyltransferase (LCAT) and solute carrier family 2 member 1 (SLC2A1) have ever been reported to be associated with adipogenesis. Interestingly, LCAT was down-regulated and expressed along with decreased levels of mRNA methylation methylation in the low-fat group. Mechanistically, the highly expressed ALKBH5 gene regulates LCAT RNA demethylation and affects LCAT mRNA stability. In addition, LCAT inhibits preadipocyte proliferation and promotes preadipocyte differentiation, and plays a key role in adipogenesis. CONCLUSIONS: In conclusion, ALKBH5 mediates RNA stability of LCAT through demethylation and affects chicken adipogenesis. This study provides a theoretical basis for further understanding of RNA methylation regulation in chicken adipogenesis.


Assuntos
Adenosina , Adipogenia , Homólogo AlkB 5 da RNA Desmetilase , Galinhas , Fosfatidilcolina-Esterol O-Aciltransferase , Estabilidade de RNA , Animais , Adipogenia/genética , Galinhas/genética , Galinhas/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Feminino , Adenosina/análogos & derivados , Adenosina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Metilação
14.
Clin Immunol ; 261: 109929, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38331303

RESUMO

Previous studies have shown that epigenetic factors are involved in the occurrence and development of rheumatoid arthritis (RA). However, the role of N6-methyladenosine (m6A) methylation in RA has not been determined. The aim of this study was to investigate the role and regulatory mechanisms of hypoxia-induced expression of the m6A demethylase alkB homolog 5 (ALKBH5) in RA fibroblast-like synoviocytes (FLSs). Synovial tissues were collected from RA and osteoarthritis (OA) patients, and RA FLSs were obtained. ALKBH5 expression in RA FLSs and collagen-induced arthritis (CIA) model rats was determined using quantitative reverse transcription-PCR (qRT-PCR), western blotting and immunohistochemistry (IHC). Using ALKBH5 overexpression and knockdown, we determined the role of ALKBH5 in RA FLS aggression and inflammation. The role of ALKBH5 in RA FLS regulation was explored using m6A-methylated RNA sequencing and methylated RNA immunoprecipitation coupled with quantitative real-time PCR. The expression of ALKBH5 was increased in RA synovial tissues, CIA model rats and RA FLSs, and a hypoxic environment increased the expression of ALKBH5 in FLSs. Increased expression of ALKBH5 promoted the proliferation and migration of RA-FLSs and inflammation. Conversely, decreased ALKBH5 expression inhibited the migration of RA-FLSs and inflammation. Mechanistically, hypoxia-induced ALKBH5 expression promoted FLS aggression and inflammation by regulating CH25H mRNA stability. Our study elucidated the functional roles of ALKBH5 and mRNA m6A methylation in RA and revealed that the HIF1α/2α-ALKBH5-CH25H pathway may be key for FLS aggression and inflammation. This study provides a novel approach for the treatment of RA by targeting the HIF1α/2α-ALKBH5-CH25H pathway.


Assuntos
Adenina/análogos & derivados , Agressão , Artrite Reumatoide , Humanos , Ratos , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Inflamação/metabolismo , Hipóxia , Fibroblastos/metabolismo , Proliferação de Células , Células Cultivadas , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo
15.
J Cell Sci ; 135(11)2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35552718

RESUMO

Establishment of the pluripotency regulatory network in somatic cells by introducing four transcription factors [octamer binding transcription factor 4 (OCT4; also known as POU5F1), sex determining region Y (SRY)-box 2 (SOX2), Kruppel-like factor 4 (KLF4) and cellular myelocytomatosis (c-MYC)] provides a promising tool for cell-based therapies in regenerative medicine. Nevertheless, the mechanisms at play when generating induced pluripotent stem cells from somatic cells are only partly understood. Here, we show that the RNA-specific N6-methyladenosine (m6A) demethylase ALKBH5 regulates somatic cell reprogramming in a stage-specific manner through its catalytic activity. Knockdown or knockout of Alkbh5 in the early reprogramming phase impairs reprogramming efficiency by reducing the proliferation rate through arresting the cells at G2/M phase and decreasing the upregulation of epithelial markers. On the other hand, ALKBH5 overexpression at the early reprogramming phase has no significant impact on reprogramming efficiency, whereas overexpression at the late phase enhances reprogramming by stabilizing Nanog transcripts, resulting in upregulated Nanog expression. Our study provides mechanistic insight into the crucial dynamic role of ALKBH5, mediated through its catalytic activity, in regulating somatic cell reprogramming at the post-transcriptional level. This article has an associated First Person interview with the first author of the paper.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Diferenciação Celular/fisiologia , Reprogramação Celular/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética
16.
Gastroenterology ; 165(2): 445-462, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37169182

RESUMO

BACKGROUND & AIMS: Immune checkpoint blockade therapy benefits only a small subset of patients with colorectal cancer (CRC), and identification of CRC-intrinsic events modulating immune checkpoint blockade efficacy is an unmet need. We found that AlkB homolog 5 (ALKBH5), an RNA N6-methyladenosine eraser, drives immunosuppression and is a molecular target to boost immune checkpoint blockade therapy in CRC. METHODS: Clinical significance of ALKBH5 was evaluated in human samples (n = 205). Function of ALKBH5 was investigated in allografts, CD34+ humanized mice, and Alkbh5 knockin mice. Immunity change was determined by means of flow cytometry, immunofluorescence, and functional investigation. Methylated RNA immunoprecipitation sequencing and RNA sequencing were used to identify ALKBH5 targets. Vesicle-like nanoparticle-encapsulated ALKBH5-small interfering RNA was constructed for targeting ALKBH5 in vivo. RESULTS: High ALKBH5 expression predicts poor prognosis in CRC. ALKBH5 induced myeloid-derived suppressor cell accumulation but reduced natural killer cells and cytotoxic CD8+ T cells to induce colorectal tumorigenesis in allografts, CD34+ humanized mice, and intestine-specific Alkbh5 knockin mice. Mechanistically, AXIN2, a Wnt suppressor, was identified as a target of ALKBH5. ALKBH5 binds and demethylates AXIN2 messenger RNA, which caused its dissociation from N6-methyladenosine reader IGF2BP1 and degradation, resulting in hyperactivated Wnt/ß-catenin. Subsequently, Wnt/ß-catenin targets, including Dickkopf-related protein 1 (DKK1) were induced by ALKBH5. ALKBH5-induced DKK1 recruited myeloid-derived suppressor cells to drive immunosuppression in CRC, and this effect was abolished by anti-DKK1 in vitro and in vivo. Finally, vesicle-like nanoparticle-encapsulated ALKBH5-small interfering RNA, or anti-DKK1 potentiated anti-PD1 treatment in suppressing CRC growth by enhancing antitumor immunity. CONCLUSIONS: This study identified an ALKBH5-N6-methyladenosine-AXIN2-Wnt-DKK1 axis in CRC, which drives immune suppression to facilitate tumorigenesis. Targeting of ALKBH5 is a promising strategy for sensitizing CRC to immunotherapy.


Assuntos
Neoplasias Colorretais , beta Catenina , Humanos , Camundongos , Animais , beta Catenina/genética , beta Catenina/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Inibidores de Checkpoint Imunológico/uso terapêutico , Carcinogênese/genética , Transformação Celular Neoplásica , RNA Interferente Pequeno/metabolismo , Imunoterapia , Terapia de Imunossupressão , Neoplasias Colorretais/terapia , Neoplasias Colorretais/tratamento farmacológico , Proteína Axina , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo
17.
Biochem Soc Trans ; 52(2): 707-717, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38629637

RESUMO

The RNA modification N6-methyladenosine (m6A) is conserved across eukaryotes, and profoundly influences RNA metabolism, including regulating RNA stability. METTL3 and METTL14, together with several accessory components, form a 'writer' complex catalysing m6A modification. Conversely, FTO and ALKBH5 function as demethylases, rendering m6A dynamic. Key to understanding the functional significance of m6A is its 'reader' proteins, exemplified by YTH-domain-containing proteins (YTHDFs) canonical reader and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) non-canonical reader. These proteins play a crucial role in determining RNA stability: YTHDFs mainly promote mRNA degradation through different cytoplasmic pathways, whereas IGF2BPs function to maintain mRNA stability. Additionally, YTHDC1 functions within the nucleus to degrade or protect certain m6A-containing RNAs, and other non-canonical readers also contribute to RNA stability regulation. Notably, m6A regulates retrotransposon LINE1 RNA stability and/or transcription via multiple mechanisms. However, conflicting observations underscore the complexities underlying m6A's regulation of RNA stability depending upon the RNA sequence/structure context, developmental stage, and/or cellular environment. Understanding the interplay between m6A and other RNA regulatory elements is pivotal in deciphering the multifaceted roles m6A plays in RNA stability regulation and broader cellular biology.


Assuntos
Adenosina , Adenosina/análogos & derivados , Estabilidade de RNA , Proteínas de Ligação a RNA , Adenosina/metabolismo , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Metiltransferases/metabolismo , RNA/metabolismo , RNA/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Processamento Pós-Transcricional do RNA , Metilação de RNA
18.
Toxicol Appl Pharmacol ; 483: 116807, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38199493

RESUMO

N6-methyladenosine (m6A) is the most prevalent mRNA modification, and it is verified to be closely correlated with cancer occurrence and progression. The m6A demethylase ALKBH5 (alkB homolog 5) is dysregulated in various cancers. However, the role and underlying mechanism of ALKBH5 in the pathogenesis and especially the chemo-resistance of non-small cell lung cancer (NSCLC) is poorly elucidated. The current study shows that ALKBH5 expression is reduced in paclitaxel (PTX) resistant NSCLC cells and down-regulation of ALKBH5 usually implies poor prognosis of NSCLC patients. Over-expression of ALKBH5 in PTX-resistant cells can suppress cell proliferation and enhance chemo-sensitivity, while knockdown of ALKBH5 exerts the opposite effect, which further supports the tumor suppressive role of ALKBH5. Over-expression of ALKBH5 can also reverse the epithelial-mesenchymal transition (EMT) process in PTX-resistant cancer cells. Mechanistically, data from RNA-seq, real-time PCR and western blotting indicate that CEMIP (cell migration inducing hyaluronidase 1), also known as KIAA1199, may be the downstream target of ALKBH5. Furthermore, ALKBH5 negatively regulates the CEMIP level by reducing the stability of CEMIP mRNA. Collectively, the current data demonstrate that the ALKBH5/CEMIP axis modulates the EMT process in NSCLC, which in turn regulates the chemo-sensitivity of cancer cells to PTX.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Paclitaxel/farmacologia , RNA Mensageiro/metabolismo
19.
J Cardiovasc Pharmacol ; 83(2): 183-192, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-37989146

RESUMO

ABSTRACT: N(6)-methyladenosine (m6A) methylation modification is involved in the progression of myocardial infarction (MI). In this study, we investigated the effects of demethylase alkylation repair homolog 5 (ALKBH5) on cell apoptosis and oxidative stress in MI. The ischemia/reperfusion (I/R) injury mouse model and hypoxia/reoxygenation (H/R) cell model were established. The levels of ALKBH5 and mitsugumin 53 (MG53) were measured by quantitative real-time polymerase chain reaction, immunohistochemical, and immunofluorescence analysis. Apoptosis was evaluated by TUNEL assay, flow cytometry, and western blot. Oxidative stress was assessed by antioxidant index kits. Methylation was analyzed by RNA binding protein immunoprecipitation (RIP), MeRIP, and dual-luciferase reporter assay. We observed that ALKBH5 and MG53 were highly expressed in MI. Overexpression of ALKBH5 inhibited H/R-induced cardiomyocyte apoptosis and oxidative stress in vitro, and it inhibited I/R-induced collagen deposition, cardiac function, and apoptosis in vivo. ALKBH5 could bind to MG53, inhibit m6A methylation of MG53, and increase its mRNA stability. Silencing of MG53 counteracted the inhibition of apoptosis and oxidative stress induced by ALKBH5. In conclusion, ALKBH5 suppressed m6A methylation of MG53 and inhibited MG53 degradation to inhibit apoptosis and oxidative stress of cardiomyocytes, thereby attenuating MI. The results provided a theoretical basis that ALKBH5 is a potential target for MI treatment.


Assuntos
Adenosina , Enzimas AlkB , Homólogo AlkB 5 da RNA Desmetilase , Infarto do Miocárdio , Estresse Oxidativo , Animais , Camundongos , Adenina/análogos & derivados , Adenosina/análogos & derivados , Enzimas AlkB/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Apoptose , Proteínas de Membrana , Metilação , Infarto do Miocárdio/metabolismo
20.
Cell Mol Biol (Noisy-le-grand) ; 70(5): 161-169, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38814199

RESUMO

Endometrial cancer (EC) is a common malignant tumor in the female reproductive system. Circular RNAs (circRNAs) and N6-methyladenosine (m6A) modification are widely involved in cancer progression. Nevertheless, the cross-talk between circ-NAB1 and m6A as well as the biological functions of circ-NAB1 in EC remain unclear. Circ-NAB1 was observed to be upregulated in EC tissues and cells by RT-qPCR. MeRIP and RNA pull-down assays were utilized for detecting the m6A modification of circ-NAB1. The interaction between circ-NAB1 and RNAs was also detected. Colony formation, transwell, flow cytometry, and western blot were utilized for measuring EC cell behaviors. Mechanically, we proved the m6A demethylase alkylation repair homolog protein 5 (ALKBH5) can mediate circ-NAB1 expression through an m6A-YTHDF2-dependent manner. Circ-NAB1 overexpression can promote cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) process, and cell cycle through functional assays. Circ-NAB1 knockdown exerts the opposite function on EC cells. Furthermore, we proved that circ-NAB1 can sponge miR-876-3p to upregulate the target gene cyclin-dependent kinase inhibitor 3 (CDKN3) in EC cells. CDKN3 overexpression can reverse the impacts of circ-NAB1 depletion on EC cell behaviors. Collectively, we proved that ALKBH5-mediated m6A modification of circ-NAB1 promoted EMT process and cell cycle in EC via targeting the miR-876-3p/CDKN3 axis.


Assuntos
Adenosina , Ciclo Celular , Proliferação de Células , Neoplasias do Endométrio , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , RNA Circular , Feminino , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Neoplasias do Endométrio/metabolismo , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética
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