RESUMEN
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.
Asunto(s)
Adenosina , Adenosina/análogos & derivados , ARN Helicasas DEAD-box , Exorribonucleasas , Inestabilidad Genómica , Metiltransferasas , Estructuras R-Loop , ARN Polimerasa II , Terminación de la Transcripción Genética , Humanos , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Adenosina/metabolismo , Adenosina/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Células HEK293 , Cromatina/metabolismo , Cromatina/genética , Daño del ADN , Células HeLa , ARN/metabolismo , ARN/genética , Transcripción Genética , Metilación de ARNRESUMEN
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.
Asunto(s)
Diferenciación Celular/genética , Diferenciación Celular/fisiología , Metiltransferasas/genética , Metiltransferasas/metabolismo , Células T Auxiliares Foliculares/metabolismo , Animales , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Centro Germinal/inmunología , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Activación de Linfocitos , Linfocitos Nulos , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , ARN Mensajero/metabolismo , Receptores CXCR5/metabolismoRESUMEN
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.