Protein Phosphatase 2ACα Regulates ATR-Mediated Endogenous DNA Damage Response Against Microcephaly.

Protein phosphatase 2A (PP2A) is an abundant heterotrimeric holoenzyme in eukaryotic cells coordinating with specific kinases to regulate spatial-temporal protein dephosphorylation in various biological processes. However, the function of PP2A in cortical neurogenesis remains largely unknown. Here, we report that neuronal-specific deletion of Pp2acα in mice displayed microcephaly, with significantly smaller brains and defective learning and memory ability. Mechanistically, neuronal Pp2acα deficiency resulted in elevated endogenous DNA damage and activation of ATR/CHK1 signaling. It was further induced by the loss of direct interaction between PP2AC and ATR as well as the function of PP2AC to dephosphorylate ATR. Importantly, ATR/CHK1 signaling dysregulation altered both the expression and activity of several critical downstream factors including P53, P21, Bcl2, and Bax, which led to decreased proliferation of cortical progenitor cells and increased apoptosis in developing cortical neurons. Taken together, our results indicate an essential function of PP2ACα in endogenous DNA damage response-mediated ATR signaling during neurogenesis, and defective PP2ACα in neurons contributes to microcephaly.

DBC1 maintains skeletal muscle integrity by enhancing myogenesis and preventing myofibre wasting.

BACKGROUND: Skeletal muscle atrophy, particularly ageing-related muscular atrophy such as sarcopenia, is a significant health concern. Despite its prevalence, the underlying mechanisms remain poorly understood, and specific approved medications are currently unavailable. Deleted in breast cancer 1 (DBC1) is a well-known regulator of senescence, metabolism or apoptosis. Recent reports suggest that DBC1 may also potentially regulate muscle function, as mice lacking DBC1 exhibit weakness and limpness. However, the function of DBC1 in skeletal muscle and its associated molecular mechanisms remain unknown, thus prompting the focus of this study. METHODS: Tibialis anterior (TA) muscle-specific DBC1 knockdown C57BL/6J male mice were generated through a single injection of 2.00 E + 11 vg of adeno-associated virus 9 delivering single-guide RNA for DBC1. Grip strength and endurance were assessed 2 months later, followed by skeletal muscle harvest. Muscle atrophy model was generated by cast immobilization of the mouse hindlimb for 2 weeks. Molecular markers of atrophy were probed in muscles upon termination. Cardiotoxin (CTX) was injected in TA muscles of DBC1 knockdown mice, and muscle regeneration was assessed by immunohistochemistry, quantitative PCR and western blotting. DBC1 knockdown C2C12 cells and myotubes were investigated using immunofluorescence staining, Seahorse, immunohistology, fluorescence-activated cell sorting and RNA-sequencing analyses. RESULTS: DBC1 knockdown in skeletal muscle of young mice led to signatures of muscle atrophy, including a 28% reduction in muscle grip force (P = 0.023), a 54.4% reduction in running distance (P = 0.002), a 14.3% reduction in muscle mass (P = 0.007) and significantly smaller myofibre cross-sectional areas (P < 0.0001). DBC1 levels decrease in age-related or limb immobilization-induced atrophic mouse muscles and overexpress DBC1-attenuated atrophic phenotypes in these mice. Muscle regeneration was hampered in mice with CTX-induced muscle injury by DBC1 knockdown, as evidenced by reductions in myofibre cross-sectional areas of regenerating myofibres with centralized nuclei (P < 0.0001), percentages of MyoG+ nuclei (P < 0.0001) and fusion index (P < 0.0001). DBC1 transcriptionally regulated mouse double minute 2 (MDM2), which mediated ubiquitination and degradation of forkhead box O3 (FOXO3). Increased FOXO3 proteins hampered myogenesis in DBC1 knockdown satellite cells by compromising around 50% of mitochondrial functions (P < 0.001) and exacerbated atrophy in DBC1 knockdown myofibres by activating the ubiquitin-proteasome and autophagy-lysosome pathways. CONCLUSIONS: DBC1 is essential in maintaining skeletal muscle integrity by protecting against myofibres wasting and enhancing muscle regeneration via FOXO3. This research highlights the significance of DBC1 for healthy skeletal muscle function and its connection to muscular atrophy.

Mic60 is essential to maintain mitochondrial integrity and to prevent encephalomyopathy.

Mitochondrial encephalomyopathies (ME) are frequently associated with mutations of mitochondrial DNA, but the pathogenesis of a subset of ME (sME) remains elusive. Here we report that haploinsufficiency of a mitochondrial inner membrane protein, Mic60, causes progressive neurological abnormalities with insulted mitochondrial structure and neuronal loss in mice. In addition, haploinsufficiency of Mic60 reduces mitochondrial membrane potential and cellular ATP production, increases reactive oxygen species, and alters mitochondrial oxidative phosphorylation complexes in neurons in an age-dependent manner. Moreover, haploinsufficiency of Mic60 compromises brain glucose intake and oxygen consumption in mice, resembling human ME syndrome. We further discover that MIC60 protein expression declined significantly in human sME, implying that insufficient MIC60 may contribute for pathogenesis of human ME. Notably, systemic administration of antioxidant N-acetylcysteine largely reverses mitochondrial dysfunctions and metabolic disorders in haplo-insufficient Mic60 mice, also restores neurological abnormal symptom. These results reveal Mic60 is required in the maintenance of mitochondrial integrity and function, and likely a potential therapeutics target for mitochondrial encephalomyopathies.

ALKBH5 in mouse testicular Sertoli cells regulates Cdh2 mRNA translation to maintain blood-testis barrier integrity.

BACKGROUND: RNA N6-methyladenosine (m6A) is involved in mammalian spermatogenesis. In both germ cells and Leydig cells, ALKBH5 regulates spermatogenesis and androgen synthesis in an m6A-dependent manner. However, it is unclear whether ALKBH5 plays a role in testicular Sertoli cells, which constitute the blood-testis barrier (BTB) through cell junctions between adjacent Sertoli cells. METHODS: ALKBH5 expression in the testes of humans and mice was detected by immunohistochemical staining and immunofluorescence staining. BTB integrity was evaluated by BTB assay. m6A-seq was performed to screen for BTB-related molecules regulated by ALKBH5. m6A immunoprecipitation-quantitative real-time polymerase chain reaction (qPCR), RNA immunoprecipitation-qPCR, western blot, coimmunoprecipitation, and polysome fractionation-qPCR analyses were performed to explore the mechanisms of ALKBH5 in BTB. Transmission electron microscopy was applied to observe the BTB ultrastructure. RESULTS: ALKBH5 in Sertoli cells is related to the integrity of the BTB. Subsequently, the m6A level on Cdh2 mRNA, encoding a structural protein N-cadherin in the BTB, was found to be regulated by ALKBH5. IGF2BP1/2/3 complexes and YTHDF1 promoted Cdh2 mRNA translation. In addition, we found that basal endoplasmic specialization, in which N-cadherin is a main structural protein, was severely disordered in the testes of Alkbh5-knockout mice. CONCLUSIONS: Our study revealed that ALKBH5 regulates BTB integrity via basal endoplasmic specialization by affecting Cdh2 mRNA translation.

METTL3 regulates m6A methylation of PTCH1 and GLI2 in Sonic hedgehog signaling to promote tumor progression in SHH-medulloblastoma.

SHH subgroup medulloblastoma (SHH-MB) is one of the most common malignant pediatric tumors that arises in the cerebellum. Previously, we showed that RNA m6A methylation participates in regulation of cerebellar development. Here we investigate whether dysregulated m6A methylation contributes to tumorigenesis of SHH-MB. We show that high expression of m6A methyltransferase METTL3 associates with worse survival in the patients with SHH-MB. A large number of hypermethylated transcripts are identified in SHH-MB tumor cells by m6A-seq. We find that METTL3 promotes tumor progression via activating Sonic hedgehog signaling. Mechanistically, METTL3 methylates PTCH1 and GLI2 RNAs and further regulates their RNA stability and translation. Importantly, targeting METTL3 by depleting METTL3 expression or treatment with its catalytic inhibitor STM2457 restrains tumor progression. Collectively, this study shows a critical function for METTL3 and m6A methylation in SHH-MB, indicative of a potential role of METTL3 as therapeutic target in SHH-MB.

m6A demethylase ALKBH5 is required for antibacterial innate defense by intrinsic motivation of neutrophil migration.

Neutrophil migration into the site of infection is necessary for antibacterial innate defense, whereas impaired neutrophil migration may result in excessive inflammation and even sepsis. The neutrophil migration directed by extracellular signals such as chemokines has been extensively studied, yet the intrinsic mechanism for determining neutrophil ability to migrate needs further investigation. N6-methyladenosine (m6A) RNA modification is important in immunity and inflammation, and our preliminary data indicate downregulation of RNA m6A demethylase alkB homolog 5 (ALKBH5) in neutrophils during bacterial infection. Whether m6A modification and ALKBH5 might intrinsically modulate neutrophil innate response remain unknown. Here we report that ALKBH5 is required for antibacterial innate defense by enhancing intrinsic ability of neutrophil migration. We found that deficiency of ALKBH5 increased mortality of mice with polymicrobial sepsis induced by cecal ligation and puncture (CLP), and Alkbh5-deficient CLP mice exhibited higher bacterial burden and massive proinflammatory cytokine production in the peritoneal cavity and blood because of less neutrophil migration. Alkbh5-deficient neutrophils had lower CXCR2 expression, thus exhibiting impaired migration toward chemokine CXCL2. Mechanistically, ALKBH5-mediated m6A demethylation empowered neutrophils with high migration capability through altering the RNA decay, consequently regulating protein expression of its targets, neutrophil migration-related molecules, including increased expression of neutrophil migration-promoting CXCR2 and NLRP12, but decreased expression of neutrophil migration-suppressive PTGER4, TNC, and WNK1. Our findings reveal a previously unknown role of ALKBH5 in imprinting migration-promoting transcriptome signatures in neutrophils and intrinsically promoting neutrophil migration for antibacterial defense, highlighting the potential application of targeting neutrophil m6A modification in controlling bacterial infections.

Rare and misincorporated DNA N6-methyladenine is a hallmark of cytotoxic stresses for selectively stimulating the stemness and proliferation of glioblastoma cells.

The entity of DNA N6-methyladenine (6mA) in mammals remains elusive and subsequently its roles in diseases are poorly understood. Here we exploited a bacterial DNA contamination-free and ultrasensitive UHPLC-MS/MS assay to reassess DNA 6mA in human glioblastomas and unveiled that DNA 6mA (~0.08 ppm) is extremely rare. By the use of two independent heavy stable isotope-labeling strategies, we further prove that the observed 6mA is solely generated by DNA polymerase-mediated misinocorporation. In vitro experiments point toward that the generation of misincorporated DNA 6mA is associated with the cellular stresses-caused release of RNA N6-methyladenine (m6A) nucleoside, which is profoundly inhibited by hypoxia milieu. Consistently, compared with normal brain tissues, DNA 6mA decreases in hypoxic human gliomas. Our data also strongly support that rare DNA 6mA rather than relatively abundant DNA 5-methylcytosine and 5-hydroxymethylcytosine is a hallmark of poor prognosis of IDH1/2 mutation-absent glioblastoma patients, reflecting the incidence of cytotoxic stresses and subsequent release of m6A nucleoside. The released m6A nucleoside may selectively preserve a subset of the glioblastoma cells and stimulate their stemness and proliferation. Noteworthily, demethylation-inhibiting IDH1 mutation increases the DNA 6mA content in human gliomas, but the depletion of the demethylase candidate ALKBH1 fails to do so, together suggesting the presence of other unknown 6mA demethylase for erasing misincorporated DNA 6mA. This is the first report on the identification of the misincorporated 6mA together with its origin and roles in diseases.

Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques.

SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Based on our findings, neuropilin 1 (NRP1), a receptor of SARS-CoV-2, was significantly elevated in cerebral cortex post infection, accompanied by active immune response releasing inflammatory factors and signal transmission among tissues, which enhanced infection of the central nervous system (CNS) in a positive feedback way, leading to viral encephalitis. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.

METTL3-mediated RNA m6A Hypermethylation Promotes Tumorigenesis and GH Secretion of Pituitary Somatotroph Adenomas.

INTRODUCTION: Pituitary growth hormone-secreting (GH) pituitary adenomas (PAs) cause mass effects and dysregulated hypersecretion of GH. However, somatic mutation burden is low in PAs. While progress has been made in identifying the epigenetic changes involved in GH-PA initiation, the precise details of its tumorigenesis in GH-PA patients remains to be elucidated. As N6-methyladenosine (m6A) has been shown to often play a critical role in various tumors, it represents a possible initiation point for the tumorigenesis of pituitary adenomas. However, the role of RNA methylation in GH adenomas remains unclear. METHODS: Protein expression of m6A regulators was measured by immunohistochemistry. Global levels and distribution of m6A methylation were separately analyzed by m6A enzyme-linked immunosorbent assay and m6A sequencing (m6A-seq). RNA interference and lentivirus knockdown system were used to investigate the role of methyltransferase-like 3 (METTL3) and its m6A- dependent regulatory mechanism in tumor progression and GH secretion. RESULTS: We show that both METTL3 messenger RNA and protein expression are elevated in GH-PA samples when compared with both normal pituitary tissue specimens and nonsecreting pituitary adenomas. Levels of m6A modification increased in GH-PAs, and hypermethylated RNAs are involved in hormone secretion and cell development. Knockdown of METTL3 in GH3 cell line resulted in decreased cell growth and GH secretion. Importantly, we found that GNAS and GADD45γ act as the downstream targets in this process. CONCLUSION: Our findings strongly suggest that m6A methyltransferase METTL3 promotes tumor growth and hormone secretion by increasing expression of GNAS and GADD45γ in a m6A-dependent manner. Thus, METTL3 and the methylated RNAs constitute suitable targets for clinical treatment of GH-PAs.

RNA m6A Demethylase ALKBH5 Protects Against Pancreatic Ductal Adenocarcinoma via Targeting Regulators of Iron Metabolism.

Although RNA m6A regulators have been implicated in the tumorigenesis of several different types of tumors, including pancreatic cancer, their clinical relevance and intrinsic regulatory mechanism remain elusive. This study analyzed eight m6A regulators (METTL3, METTL14, WTAP, FTO, ALKBH5, and YTHDF1-3) in pancreatic ductal adenocarcinoma (PDAC) and found that only RNA m6A demethylase ALKBH5 serves as an independent favorable prognostic marker for this tumor. To better understand the molecular mechanism underlying the protective effect conferred by ALKBH5 against pancreatic tumorigenesis, we performed a transcriptome-wide analysis of m6A methylation, gene expression, and alternative splicing (AS) using the MIA PaCa-2 stable cell line with ALKBH5 overexpression. We demonstrated that ALKBH5 overexpression induced a reduction in RNA m6A levels globally. Furthermore, mRNAs encoding ubiquitin ligase FBXL5, and mitochondrial iron importers SLC25A28 and SLC25A37, were identified as substrates of ALKBH5. Mechanistically, the RNA stabilities of FBXL5 and SLC25A28, and the AS of SLC25A37 were affected, which led to their upregulation in pancreatic cancer cell line. Particularly, we observed that downregulation of FBXL5 in tumor samples correlated with shorter survival time of patients. Owing to FBXL5-mediated degradation, ALKBH5 overexpression incurred a significant reduction in iron-regulatory protein IRP2 and the modulator of epithelial-mesenchymal transition (EMT) SNAI1. Notably, ALKBH5 overexpression led to a significant reduction in intracellular iron levels as well as cell migratory and invasive abilities, which could be rescued by knocking down FBXL5. Overall, our results reveal a previously uncharacterized mechanism of ALKBH5 in protecting against PDAC through modulating regulators of iron metabolism and underscore the multifaceted role of m6A in pancreatic cancer.

RNA N6-methyladenosine modification, spermatogenesis, and human male infertility.

RNA N6-methyladenosine (m6A) modification is one of the main forms of posttranscriptional modification, and its dysregulation is involved in a series of pathological processes. RNA m6A regulators, which mediate dynamic RNA m6A modification, are expressed in almost all types of testicular cells, including spermatogenetic cells and somatic cells. Cumulative studies have found that knockout of RNA m6A regulators in the testis leads to abnormal metabolism of the target mRNAs, which eventually causes spermatogenetic disorders and infertility. To date, a role for dysregulated RNA m6A modification in human male infertility remains elusive; however, dysregulated expression of RNA m6A regulators in abnormal human semen samples, including oligospermia, asthenozoospermia and azoospermia, has been found. Therefore, we speculate that abnormal RNA m6A methylation may be an important mechanism of male infertility. In this review, we summarize the recent findings regarding the spatiotemporal expression of RNA m6A regulators in the testes, mechanisms of RNA m6A modification in spermatogenesis and the relation between dysregulated RNA m6A regulators and human male infertility. In addition, we also discuss future directions in studying the molecular mechanism of male infertility and exploring their clinical applications from the viewpoint of RNA m6A modification.

Genome-wide 5-Hydroxymethylcytosine Profiling Analysis Identifies MAP7D1 as A Novel Regulator of Lymph Node Metastasis in Breast Cancer.

Although DNA 5-hydroxymethylcytosine (5hmC) is recognized as an important epigenetic mark in cancer, its precise role in lymph node metastasis remains elusive. In this study, we investigated how 5hmC associates with lymph node metastasis in breast cancer. Accompanying with high expression of TET1 and TET2 proteins, large numbers of genes in the metastasis-positive primary tumors exhibit higher 5hmC levels than those in the metastasis-negative primary tumors. In contrast, the TET protein expression and DNA 5hmC decrease significantly within the metastatic lesions in the lymph nodes compared to those in their matched primary tumors. Through genome-wide analysis of 8 sets of primary tumors, we identified 100 high-confidence metastasis-associated 5hmC signatures, and it is found that increased levels of DNA 5hmC and gene expression of MAP7D1 associate with high risk of lymph node metastasis. Furthermore, we demonstrate that MAP7D1, regulated by TET1, promotes tumor growth and metastasis. In conclusion, the dynamic 5hmC profiles during lymph node metastasis suggest a link between DNA 5hmC and lymph node metastasis. Meanwhile, the role of MAP7D1 in breast cancer progression suggests that the metastasis-associated 5hmC signatures are potential biomarkers to predict the risk for lymph node metastasis, which may serve as diagnostic and therapeutic targets for metastatic breast cancer.

Loss of 5-Hydroxymethylcytosine as an Epigenetic Signature That Correlates With Poor Outcomes in Patients With Medulloblastoma.

Medulloblastoma, as the most common malignant brain tumor in children, exhibits highly dysregulated DNA methylation. The novel epigenetic marker-5-hydroxymethylcytosine (5hmC) plays essential role in gene regulation during brain development and in brain tumors. However, the biological and clinical implications of 5hmC in medulloblastoma are still unclear. Here, we detected global 5hmC levels in two independent medulloblastoma patient cohorts (discovery cohort: n = 81; validation cohort: n = 171) using ultra-high performance liquid chromatography-tandem mass spectrometry analysis. Immunohistochemistry was used to identify the cell proliferation and expression of Ten-eleven translocation 1 and 2 (TET1/2). The prognostic impacts of covariates on progression-free survival (PFS) and overall survival (OS) were evaluated using multivariate Cox hazards regression models. We observed that global 5hmC levels were decreased in medulloblastomas compared to normal cerebellums (P < 0.001). Multivariate analysis showed that low global 5hmC levels correlated with poor PFS and OS rates (discovery cohort: PFS: P = 0.003, OS: P = 0.002; validation cohort: PFS: P = 0.0002, OS: P = 0.001). Immunohistochemistry showed an inverse correlation between 5hmC score and Ki-67 index (r = -0.747, P < 0.0001). Moreover, 5hmC score in MB samples was associated with nuclear expression of TET1 (r = -0.419, P = 0.003) and TET2 (r = -0.399, P = 0.005) proteins. Our study demonstrates that loss of 5hmC is an epigenetic biomarker in medulloblastomas. Our results indicate that 5hmC could be a candidate prognostic indicator for improving survival prediction of risk stratification in patients with medulloblastoma.

Changes in DNA 5-Hydroxymethylcytosine Levels and the Underlying Mechanism in Non-functioning Pituitary Adenomas.

Epigenetic factors have been proven to contribute to pituitary adenoma formation. 5-hydroxymethylcytosine (5hmC), which is catalyzed by ten-eleven translocation 2 (TET2), is related to DNA demethylation. In order to explore the pathogenesis of non-functioning pituitary adenomas (NFPAs), we detected genomic 5hmC levels in 57 NFPAs and 5 normal pituitary glands, and TET2 expression, distribution and TET2 alteration. Genomic 5hmC levels in NFPAs were significantly lower than those in normal pituitary glands (0.38‰ (0.24‰, 0.61‰) vs. 2.47‰ (1.56‰, 2.83‰), P < 0.0001). There was positive correlation of 5hmC levels with TET2 total and nuclear expression in NFPAs (r = 0.461, P = 0.018; r = 0.458, P = 0.019). Genomic 5hmC levels in NFPAs with TET2 p.P29R were significantly lower than those in wild type NFPAs (0.33 ± 0.18‰ vs. 0.51 ± 0.25‰, P = 0.021). We found genomic 5hmC loss in human NFPAs for the first time. Genomic 5hmC levels may be affected by TET2 expression, subcellular localization and TET2 mutation.

Characterization of global 5-hydroxymethylcytosine in pediatric posterior fossa ependymoma.

BACKGROUND: 5-Hydroxymethylcytosine (5hmC) is a novel epigenetic mark and may be involved in the mechanisms of tumorigenesis and malignant transformation. However, the role of 5hmC in ependymoma, the third most common brain tumor in children, remains unclear. The aim of this study sought to identify the characterization of 5hmC levels in pediatric posterior fossa ependymoma and to evaluate whether 5hmC levels could be a potential factor to predict clinical outcomes. RESULTS: Our results showed that 5hmC levels were globally decreased in posterior fossa ependymoma compared with normal cerebellum tissues (P < 0.001). Group A posterior fossa ependymomas had higher 5hmC levels than group B tumors (P = 0.007). Moreover, 5hmC levels positively correlated with Ki-67 index in posterior fossa ependymoma (r = 0.428, P = 0.003). Multivariate Cox hazards model revealed that patients with high 5hmC levels (> 0.102%) had worse PFS and OS than patients with lower 5hmC levels (< 0.102%) (PFS: HR = 3.014; 95% CI, 1.040-8.738; P = 0.042; OS: HR = 2.788; 95% CI, 0.974-7.982; P = 0.047). CONCLUSIONS: Our findings suggest that loss of 5hmC is an epigenetic hallmark for pediatric posterior fossa ependymoma. 5hmC levels may represent a potential biomarker to predict prognosis in children with posterior fossa ependymoma.

RNA m6A Methylation Regulators Subclassify Luminal Subtype in Breast Cancer.

RNA N6-methyladenosine (m6A) methylation is the most prevalent epitranscriptomic modification in mammals, with a complex and fine-tuning regulatory system. Recent studies have illuminated the potential of m6A regulators in clinical applications including diagnosis, therapeutics, and prognosis. Based on six datasets of breast cancer in The Cancer Genome Atlas (TCGA) database and two additional proteomic datasets, we provide a comprehensive view of all the known m6A regulators in their gene expression, copy number variations (CNVs), DNA methylation status, and protein levels in breast tumors and their association with prognosis. Among four breast cancer subtypes, basal-like subtype exhibits distinct expression and genomic alteration in m6A regulators from other subtypes. Accordingly, four representative regulators (IGF2BP2, IGF2BP3, YTHDC2, and RBM15) are identified as basal-like subtype-featured genes. Notably, luminal A/B samples are subclassified into two clusters based on the methylation status of those four genes. In line with its similarity to basal-like subtype, cluster1 shows upregulation in immune-related genes and cell adhesion molecules, as well as an increased number of tumor-infiltrating lymphocytes. Besides, cluster1 has worse disease-free and progression-free survival, especially among patients diagnosed with stage II and luminal B subtype. Together, this study highlights the potential functions of m6A regulators in the occurrence and malignancy progression of breast cancer. Given the heterogeneity within luminal subtype and high risk of recurrence and metastasis in a portion of patients, the prognostic stratification of luminal A/B subtypes utilizing basal-featured m6A regulators may help to improve the accuracy of diagnosis and therapeutics of breast cancer.

N 6-methyladenosine RNA modification-mediated cellular metabolism rewiring inhibits viral replication.

Host cell metabolism can be modulated by viral infection, affecting viral survival or clearance. Yet the cellular metabolism rewiring mediated by the N 6-methyladenosine (m6A) modification in interactions between virus and host remains largely unknown. Here we report that in response to viral infection, host cells impair the enzymatic activity of the RNA m6A demethylase ALKBH5. This behavior increases the m6A methylation on α-ketoglutarate dehydrogenase (OGDH) messenger RNA (mRNA) to reduce its mRNA stability and protein expression. Reduced OGDH decreases the production of the metabolite itaconate that is required for viral replication. With reduced OGDH and itaconate production in vivo, Alkbh5-deficient mice display innate immune response-independent resistance to viral exposure. Our findings reveal that m6A RNA modification-mediated down-regulation of the OGDH-itaconate pathway reprograms cellular metabolism to inhibit viral replication, proposing potential targets for controlling viral infection.

RNA m6A methylation participates in regulation of postnatal development of the mouse cerebellum.

BACKGROUND: N6-methyladenosine (m6A) is an important epitranscriptomic mark with high abundance in the brain. Recently, it has been found to be involved in the regulation of memory formation and mammalian cortical neurogenesis. However, while it is now established that m6A methylation occurs in a spatially restricted manner, its functions in specific brain regions still await elucidation. RESULTS: We identify widespread and dynamic RNA m6A methylation in the developing mouse cerebellum and further uncover distinct features of continuous and temporal-specific m6A methylation across the four postnatal developmental processes. Temporal-specific m6A peaks from P7 to P60 exhibit remarkable changes in their distribution patterns along the mRNA transcripts. We also show spatiotemporal-specific expression of m6A writers METTL3, METTL14, and WTAP and erasers ALKBH5 and FTO in the mouse cerebellum. Ectopic expression of METTL3 mediated by lentivirus infection leads to disorganized structure of both Purkinje and glial cells. In addition, under hypobaric hypoxia exposure, Alkbh5-deletion causes abnormal cell proliferation and differentiation in the cerebellum through disturbing the balance of RNA m6A methylation in different cell fate determination genes. Notably, nuclear export of the hypermethylated RNAs is enhanced in the cerebellum of Alkbh5-deficient mice exposed to hypobaric hypoxia. CONCLUSIONS: Together, our findings provide strong evidence that RNA m6A methylation is controlled in a precise spatiotemporal manner and participates in the regulation of postnatal development of the mouse cerebellum.