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Title: METTL3-mediated m6A modification is required for cerebellar development}
Article:
Abstract:
N6-methyladenosine( m6A) RNA methylation is the most abundant modification on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex including methyltransferase-like 3( METTL3) as a key factor. However, the in vivo functions of METTL3 and m6A modification in mammalian development remain unclear. Here, we show that specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3 conditional knockout( cKO) mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of newborn cerebellar granule cells( CGCs) in the external granular layer( EGL). METTL3 depletion–induced loss of m6A modification causes extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A in regulating the development of mammalian cerebellum.
Introduction:
Multiple layers of epigenetic modifications play essential roles in neuronal development and brain function in mammals through highly coordinated epigenetic regulatory mechanisms[1–5], such as DNA methylation and demethylation[6–8], histone modifications[9, 10], and noncoding RNAs[11–13]. Additionally, RNA modifications have recently been recognized as a new layer of epigenetic regulation, among which N6-methyladenosine( m6A) is the one being most extensively studied. m6A formation is catalyzed by a methyltransferase complex including methyltransferase-like 3( METTL3), methyltransferase-like 14( METTL14), and Wilms’ tumor 1-associating protein( WTAP), among which METTL3 functions as the catalytic subunit[14, 15]. As the most abundant and reversible modification on mRNAs, m6A has been proven to play key roles in regulating RNA stability[16] and RNA splicing[17], as well as mRNA translation efficiency[18–22]. Many essential biological processes are known to be regulated by m6A, including cell fate determination[23, 24] and embryonic development[24, 25, 26]. In neuronal systems, m6A has been shown to be a dynamic modification and increases in adulthood[27], suggesting a potential function in neural plasticity and brain function. Previous studies have shown that conventional knockout of Mettl3 in mice leads to early embryonic lethality[24], and specific depletion of m6A in the nervous system by conditional knockout of Mettl14 results in extended cell cycle and altered neurogenesis in embryonic mouse cortex, through regulating the decay of neurogenesis-related transcripts[28]. Furthermore, loss of m6A eraser, the fat mass and obesity-associated protein( FTO), leads to impaired adult neurogenesis and cognition ability[29]. FTO is enriched in axons, and specifically silencing axonal FTO inhibits axon elongation by suppressing growth associated protein 43( GAP-43)[30]. Nerve lesion induces the elevation of m6A level, and loss of METTL14 or m6A binding protein YTH N6-methyladenosine RNA binding protein 1( YTHDF1) inhibits axonal regeneration[31]. All these studies indicate the important roles of m6A signaling in neuronal development and neurogenesis. The cerebellum offers an ideal model to study neurogenesis, as it contains a limited number of neuronal categories, and different types of cerebellar neurons are generated in a timely order, precisely scheduled by progenitors. The developing cerebellum consists of the external granular layer( EGL), molecular layer( ML), Purkinje cell layer( PCL), and internal granular layer( IGL)[32, 33]. It has been shown that cerebellar granule cell progenitors( GCPs) proliferate in EGL, then migrate through ML to IGL to differentiate into granule cells, generating parallel fibers and forming synaptic connections with Purkinje cells[33]. The EGL, as the exclusive source for cerebellar granule cells( CGCs)[32, 34], gradually becomes thinner and disappears around 21 days postpartum( P21) in mice[33]. Although many studies have indicated the function of epigenetic modifications in cerebellar development, little is known about the roles of m6A signaling in the development of the cerebellum and its associated behavioral phenotype. In the present work, we specifically inactivated Mettl3 in the developing mouse brain using the Nestin-Cre mediated Mettl3 conditional knockout model. Depletion of METTL3 in the brain caused severe developmental defects in both the cortical and cerebellar regions. Further analysis demonstrated drastic apoptosis of newborn CGCs, which is partially responsible for the severe cerebellar hypoplasia and a series of ataxia-like movement disorders. Furthermore, loss of m6A by inactivation of Mettl3 led to extended half-lives of mRNAs from cerebellar development- and apoptosis-associated genes, as well as aberrant mRNA splicing events on synapse-associated genes, which consequently induced inappropriate cell differentiation and cell death, indicating that METTL3-mediated m6A serves as a key regulator in modulating the development of the central nervous system at the posttranscriptional level in mammals.
Discussion:
It is well established that epigenetic machineries play essential roles in controlling the timing and linage commitment of NSCs during neurogenesis[49, 50]. Epigenetic mechanisms such as DNA methylation and histone modifications regulate brain development and plasticity at the transcriptional level[4, 51]. Additionally, noncoding RNAs and RNA modifications also appeared to add complexities at the epitranscriptomic level in regulating neuronal development and brain function[26, 52–57], and multiple layers of epigenetic modification regulations have been shown to be required for neurogenesis in the developing cerebellum[58–60]. DNA demethylation is highly active during early cerebellum development, and inhibition of DNA demethylation suppresses the circuit formation of developing granular cells[60]. GCPs and glial cells express high levels of histone deacetylase 1( HDAC1), indicating the potential role of histone modification in neurogenesis of the cerebellum[61]. The microRNAs( miRNAs) have been reported to regulate components of Hedgehog-Patched signaling in normal or transformed GCPs[62]. In addition, alternative transcription combined with alternative splicing gives rise to transcriptomic diversity during cerebellar development[59]. Recent study has reported that specific depletion of m6A in the mouse brain by conditional knockout of Mettl14 causes postponed neurogenesis in the cortex[28]. In our study, we found remarkable cerebellar developmental defects in Mettl3 conditional knockout mouse model, indicating the indispensable roles of METTL14- and METTL3-mediated m6A signaling in neuronal development. In addition, we recognized a severe cerebellar hypoplasia phenotype in Mettl3 cKO mice. Depletion of m6A caused apoptosis of the newborn granule cells, which then resulted in altered Purkinje cell and Bergmann glia architecture. Our findings demonstrated, for the first time, the regulatory function of m6A modification in cerebellar development. The development of the mouse cerebellum is quite distinct from that of the cerebral region of the brain. Neurogenesis dominantly occurs in the embryonic stage in the cortex, while in the cerebellar region, this process mainly occurs in the postnatal 2 wk[32]. A whole transcriptome-wide m6A methylation analysis depicted a spatial-specific methylation profile of mouse brain and revealed that m6A methylation had a higher level in the cerebellum than that in the cerebral cortex[35], indicating pivotal roles of m6A in this brain region. The development of the mouse cerebellum is a highly organized process involving a cooperated set of genetic and epigenetic regulations. By high throughput RNA-seq and m6A-seq, we revealed that key developmental genes like Atoh1 and Cxcr4 were abnormally up-regulated because of the extended mRNA half-lives induced by m6A depletion in the cKO mice. During the development of CGCs, apoptosis occurs occasionally as a normal process[63], whereas in the cKO mice, in addition to developmental associated genes, we also detected up-regulation of apoptosis-associated genes like Dapk1 and Fadd, with extended half-lives of their mRNAs. Hence the m6A modification on mRNAs may play an indispensable role in balancing cell survival and apoptosis by regulating mRNA stability of development- and apoptosis-associated genes. Comparison of the mouse brain m6A-seq data with the mouse synaptic proteome revealed that 76. 8% of mouse postsynaptic genes and 30% of presynaptic genes were detected with m6A RNA methylation[35]. In addition to the resistance of decay in the developmental- and apoptosis-associated genes during the cell fate conversion from GCPs to CGCs, we also recognized a set of synapse-related genes that are highly enriched in the abnormally spliced genes when m6A is depleted in the cerebellum. Grin1 had been reported to be associated with CGC survival and contribute to the survival-promoting effect of NMDA receptors in cultured CGCs[64]. In this study, we demonstrated that the altered splicing of Grin1 induced by m6A depletion resulted in excessive calcium influx and finally caused the apoptosis of the CGCs. The regulatory function of METTL3-mediated m6A in alternative splicing of synapse-associated genes implied its pivotal role in neuronal survival, connection, and maturation. Although it has been reported that cytoplasmic translocation of METTL3 alone could promote protein translation independent of its m6A activity[65], our current study demonstrated that m6A depletion induced by Mettl3 inactivation accounts for both the elevated RNA stability of development- and apoptosis-associated genes( such as Atoh1 and Dapk1) and the altered splicing of synapse-associated genes( such as Grin1), which consequently led to the severe cerebellum development defects manifested by massive apoptosis and impaired differentiation of CGCs in the knockout mouse model. Thus, it is highly unlikely that the functional role of Mettl3 in the mouse cerebellum development is m6A independent. Contrary to a previous report showing that METTL3 is widely expressed in various regions of the mammalian brain[35], we detected relatively weak METTL3 signals in the proliferating GCPs in the outer EGL of the developing mouse cerebellum by Immunohistochemistry( IHC)( S4B Fig), which might be a likely reason to explain why the proliferation of the GCPs was not substantially affected by depletion of METTL3. However, we detected dramatically elevated expression of METTL3 when GCPs differentiated into the newborn CGCs in the inner layer of EGL( S4B Fig). We highly speculate that enhanced expression of METTL3 is a pivotal event during the differentiation of CGCs. Taken together, we conclude that METTL3 is an essential regulator of cerebellar neurogenesis and development, and the transcriptome-wide gene expression dysregulation induced by m6A depletion in the absence of METTL3 finally results in CGCs apoptosis and cerebellum hypoplasia( Fig 6G).
N6-methyladenosine ( m6A ) is an abundant modification in mRNA molecules and regulates mRNA metabolism and various biological processes , such as cell fate control , early embryonic development , sex determination , and diseases like diabetes and obesity . Adenosine methylation is regulated by a large methyltransferase complex and by demethylases , as well as by other binding proteins . METTL3 is one of the core subunits of the methyltransferase complex catalyzing m6A formation . However , the role of METTL3-mediated m6A in mammalian brain development remains unclear mainly because of the lack of specific spatiotemporal knockout animal models , as conventional METTL3 knockout in mice leads to early embryonic death . In this study , we specifically inactivated METTL3 in the developing mouse brain . We detected a drastic depletion of m6A accompanied by severe developmental defects in the cerebellum of these mice . Further analysis established that METTL3-mediated m6A participates in cerebellar development by controlling mRNA stability of genes related to cerebellar development and apoptosis and by regulating alternative splicing of pre-mRNAs of synapse-associated genes .