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. 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. 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.

RNA modification meets immune metabolism: N ⁶ -methyladenosine (m ⁶ A) RNA modification regulates various cellular functions. Liu et al. found that host cells impair RNA m ⁶ A demethylase activity after viral infection, leading to increased m ⁶ A and reduced stability of α-ketoglutarate dehydrogenase (OGDH) mRNA. As a result, reduced OGDH decreases the generation of itaconate, thereby inhibiting viral replication. The authors explore the function of OGDH and itaconate in viral infection, provide insights into m ⁶ A RNA modification and metabolic reprogramming in modulating virus-host interaction, and suggest potential therapeutic targets for the control of viral infection. Science , this issue p. 1171

N ⁶ -methyladenosine (m ⁶ A) is the most abundant epitranscriptomic mark found on mRNA and has important roles in various physiological processes. Despite the relatively high m ⁶ A levels in the brain, its potential functions in the brain remain largely unexplored. We performed a transcriptome-wide methylation analysis using the mouse brain to depict its region-specific methylation profile. RNA methylation levels in mouse cerebellum are generally higher than those in the cerebral cortex. Heterogeneity of RNA methylation exists across different brain regions and different types of neural cells including the mRNAs to be methylated, their methylation levels and methylation site selection. Common and region-specific methylation have different preferences for methylation site selection and thereby different impacts on their biological functions. In addition, high methylation levels of fragile X mental retardation protein (FMRP) target mRNAs suggest that m ⁶ A methylation is likely to be used for selective recognition of target mRNAs by FMRP in the synapse. Overall, we provide a region-specific map of RNA m ⁶ A methylation and characterize the distinct features of specific and common methylation in mouse cerebellum and cerebral cortex. Our results imply that RNA m ⁶ A methylation is a newly identified element in the region-specific gene regulatory network in the mouse brain.

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.

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.

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.