ABSTRACT
N6-methyladenosine (m6A) is a crucial RNA modification that regulates diverse biological processes in human cells, but its co-transcriptional deposition and functions remain poorly understood. Here, we identified the RNA helicase DDX21 with a previously unrecognized role in directing m6A modification on nascent RNA for co-transcriptional regulation. DDX21 interacts with METTL3 for co-recruitment to chromatin through its recognition of R-loops, which can be formed co-transcriptionally as nascent transcripts hybridize onto the template DNA strand. Moreover, DDX21's helicase activity is needed for METTL3-mediated m6A deposition onto nascent RNA following recruitment. At transcription termination regions, this nexus of actions promotes XRN2-mediated termination of RNAPII transcription. Disruption of any of these steps, including the loss of DDX21, METTL3, or their enzymatic activities, leads to defective termination that can induce DNA damage. Therefore, we propose that the R-loop-DDX21-METTL3 nexus forges the missing link for co-transcriptional modification of m6A, coordinating transcription termination and genome stability.
Subject(s)
Adenosine , Adenosine/analogs & derivatives , DEAD-box RNA Helicases , Exoribonucleases , Genomic Instability , Methyltransferases , R-Loop Structures , RNA Polymerase II , Transcription Termination, Genetic , Humans , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Methyltransferases/metabolism , Methyltransferases/genetics , Adenosine/metabolism , Adenosine/genetics , Exoribonucleases/metabolism , Exoribonucleases/genetics , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , HEK293 Cells , Chromatin/metabolism , Chromatin/genetics , DNA Damage , HeLa Cells , RNA/metabolism , RNA/genetics , Transcription, Genetic , RNA MethylationABSTRACT
T follicular helper (TFH) cells are specialized effector CD4+ T cells critical to humoral immunity. Whether post-transcriptional regulation has a function in TFH cells is unknown. Here, we show conditional deletion of METTL3 (a methyltransferase catalyzing mRNA N6-methyladenosine (m6A) modification) in CD4+ T cells impairs TFH differentiation and germinal center responses in a cell-intrinsic manner in mice. METTL3 is necessary for expression of important TFH signature genes, including Tcf7, Bcl6, Icos and Cxcr5 and these effects depend on intact methyltransferase activity. m6A-miCLIP-seq shows the 3' UTR of Tcf7 mRNA is subjected to METTL3-dependent m6A modification. Loss of METTL3 or mutation of the Tcf7 3' UTR m6A site results in accelerated decay of Tcf7 transcripts. Importantly, ectopic expression of TCF-1 (encoded by Tcf7) rectifies TFH defects owing to METTL3 deficiency. Our findings indicate that METTL3 stabilizes Tcf7 transcripts via m6A modification to ensure activation of a TFH transcriptional program, indicating a pivotal function of post-transcriptional regulation in promoting TFH cell differentiation.
Subject(s)
Cell Differentiation/genetics , Cell Differentiation/physiology , Methyltransferases/genetics , Methyltransferases/metabolism , T Follicular Helper Cells/metabolism , Animals , Gene Expression Regulation , Gene Knockdown Techniques , Germinal Center/immunology , Hepatocyte Nuclear Factor 1-alpha/metabolism , Lymphocyte Activation , Lymphocytes, Null , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-bcl-6/metabolism , RNA, Messenger/metabolism , Receptors, CXCR5/metabolismABSTRACT
BACKGROUND: Ginkgo biloba extract is widely studied for antiaging activities, but little is known about its antiaging mechanism of protein carbonylation. In order to verify carbonyl toxification (stress) hypothesis of aging, we have investigated the effects of EGb761 on hippocampal neuronal injury and carbonyl stress of aging rats. METHODS: Seventy-two Wister male rats were randomly assigned into six groups (n = 12), normal control (NC), model control (MC), vitamin E (VE, 60 mg/kg) group, EGb761 low doses (GBEL, 8.75 mg/kg), EGb761 moderate doses (GBEM, 17.5 mg/kg), and EGb761 high doses (GBEH, 35 mg/kg). Except the NC, the other groups were subject to subcutaneous administration of 0.5% D-gal (10 ml/kg/day) for 6 weeks to induce aging model. The study detected cognitive impairment in rats by Morris water maze test and the contents of superoxidase dismutase (SOD), malondialdehyde (MDA), total antioxidant capacity (T-AOC) by the related kits. The level of 4-hydroxy-2-nonenal (4-HNE) protein adducts in rat brain was detected, and the ultrastructure of hippocampus was observed. RESULTS: The EGb761 treatment groups significantly improved the spatial learning and memory of rats. Moreover, EGb761 treatment could reduce hippocampal neuronal damage based on histopathological and ultrastructural observation. Further studies have proved that these activities are remarkably related with the reducing level of MDA, protein carbonyl and lipofuscin, and 4-HNE protein expression, as well as the increasing of SOD and T-AOC content. Furthermore, EGb761 improves telomerase activity by detecting telomerase activity in the brain of aging rats. CONCLUSION: Our data indicate that EGb761 is an effective agent against D-gal-induced hippocampal neuronal loss owing to its antioxidative as well as carbonyl stress properties. Meanwhile, the carbonylation hypothesis is confirmed that the high level of 4-HNE may cause age-related neurodegenerative disorders.
ABSTRACT
N6-methyladenosine (m6A), catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14, is the most abundant RNA modification in mRNAs and participates in diverse biological processes. However, the roles and precise mechanisms of m6A modification in regulating neuronal development and adult neurogenesis remain unclear. Here, we examined the function of Mettl3, the key component of the complex, in neuronal development and adult neurogenesis of mice. We found that the depletion of Mettl3 significantly reduced m6A levels in adult neural stem cells (aNSCs) and inhibited the proliferation of aNSCs. Mettl3 depletion not only inhibited neuronal development and skewed the differentiation of aNSCs more toward glial lineage, but also affected the morphological maturation of newborn neurons in the adult brain. m6A immunoprecipitation combined with deep sequencing (MeRIP-seq) revealed that m6A was predominantly enriched in transcripts related to neurogenesis and neuronal development. Mechanistically, m6A was present on the transcripts of histone methyltransferase Ezh2, and its reduction upon Mettl3 knockdown decreased both Ezh2 protein expression and consequent H3K27me3 levels. The defects of neurogenesis and neuronal development induced by Mettl3 depletion could be rescued by Ezh2 overexpression. Collectively, our results uncover a crosstalk between RNA and histone modifications and indicate that Mettl3-mediated m6A modification plays an important role in regulating neurogenesis and neuronal development through modulating Ezh2.