RESEARCH ARTICLE METTL3-mediated m6A modification is required for cerebellar development Chen-Xin Wang1,2☯, Guan-Shen Cui3,4☯, Xiuying Liu5☯, Kai Xu1,2☯, Meng Wang5☯, Xin- Xin Zhang1, Li-Yuan Jiang1, Ang Li2,3, Ying Yang3, Wei-Yi Lai2,6, Bao-Fa Sun2,3,7, Gui- Bin Jiang6, Hai-Lin Wang6, Wei-Min Tong8, Wei Li1,2,7, Xiu-Jie Wang2,5,7*, Yun- Gui Yang2,3,7*, Qi Zhou1,2,7* 1 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China, 2 University of Chinese Academy of Sciences, Beijing, China, 3 Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing a1111111111 Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 4 Sino-Danish College, University of a1111111111 Chinese Academy of Sciences, Beijing, China, 5 Key Laboratory of Genetic Network Biology, Institute of a1111111111 Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China, 6 State Key Laboratory a1111111111 of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese a1111111111 Academy of Sciences, Beijing, China, 7 Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China, 8 Department of Pathology, Center for Experimental Animal Research, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China ☯ These authors contributed equally to this work. OPEN ACCESS * [email protected] (XJW); [email protected] (YGY); [email protected] (QZ) Citation: Wang C-X, Cui G-S, Liu X, Xu K, Wang M, Zhang X-X, et al. (2018) METTL3-mediated m6A modification is required for cerebellar Abstract development. PLoS Biol 16(6): e2004880. https:// doi.org/10.1371/journal.pbio.2004880 N6-methyladenosine (m6A) RNA methylation is the most abundant modification on mRNAs 6 Academic Editor: Chuan He, The University of and plays important roles in various biological processes. The formation of m A is catalyzed Chicago, United States of America by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key fac- 6 Received: November 21, 2017 tor. However, the in vivo functions of METTL3 and m A modification in mammalian develop- ment remain unclear. Here, we show that specific inactivation of Mettl3 in mouse nervous Accepted: May 15, 2018 system causes severe developmental defects in the brain. Mettl3 conditional knockout Published: June 7, 2018 (cKO) mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of Copyright: © 2018 Wang et al. This is an open newborn cerebellar granule cells (CGCs) in the external granular layer (EGL). METTL3 access article distributed under the terms of the depletion±induced loss of m6A modification causes extended RNA half-lives and aberrant Creative Commons Attribution License, which permits unrestricted use, distribution, and splicing events, consequently leading to dysregulation of transcriptome-wide gene expres- reproduction in any medium, provided the original sion and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A author and source are credited. in regulating the development of mammalian cerebellum. Data Availability Statement: All relevant data are within the paper and its Supporting information files, except that the sequencing data have been deposited in NCBI GEO database (https://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= Author summary GSE100528) and can be accessed via accession 6-methyladenosine (m6A) is an abundant modification in mRNA molecules and regu- number GSE100528. N lates mRNA metabolism and various biological processes, such as cell fate control, early Funding: National Natural Science Foundation of embryonic development, sex determination, and diseases like diabetes and obesity. Aden- China https://isisn.nsfc.gov.cn/egrantindex/ osine methylation is regulated by a large methyltransferase complex and by demethylases, funcindex/prjsearch-list (grant number 91540204). Received by X. -J. W. The funder had no role in as well as by other binding proteins. METTL3 is one of the core subunits of the methyl- 6 study design, data collection and analysis, decision transferase complex catalyzing m A formation. However, the role of METTL3-mediated PLOS Biology | https://doi.org/10.1371/journal.pbio.2004880 June 7, 2018 1 / 29 METTL3 is required for mammalian cerebellar development to publish, or preparation of the manuscript. National Natural Science Foundation of China m6A in mammalian brain development remains unclear mainly because of the lack of spe- https://isisn.nsfc.gov.cn/egrantindex/funcindex/ cific spatiotemporal knockout animal models, as conventional METTL3 knockout in mice prjsearch-list (grant number 31430022). Received leads to early embryonic death. In this study, we specifically inactivated METTL3 in the by Y. -G. Y. The funder had no role in study design, 6 data collection and analysis, decision to publish, or developing mouse brain. We detected a drastic depletion of m A accompanied by severe preparation of the manuscript. National Natural developmental defects in the cerebellum of these mice. Further analysis established that 6 Science Foundation of China https://isisn.nsfc.gov. METTL3-mediated m A participates in cerebellar development by controlling mRNA sta- cn/egrantindex/funcindex/prjsearch-list (grant bility of genes related to cerebellar development and apoptosis and by regulating alterna- number 31471395). Received by Q. Z. The funder tive splicing of pre-mRNAs of synapse-associated genes. had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Natural Science Foundation of China https://isisn.nsfc.gov.cn/egrantindex/ funcindex/prjsearch-list (grant number 31625016). Introduction Received by Y. -G. Y. The funder had no role in Multiple layers of epigenetic modifications play essential roles in neuronal development and study design, data collection and analysis, decision to publish, or preparation of the manuscript. brain function in mammals through highly coordinated epigenetic regulatory mechanisms [1± National Natural Science Foundation of China 5], such as DNA methylation and demethylation [6±8], histone modifications [9, 10], and non- https://isisn.nsfc.gov.cn/egrantindex/funcindex/ coding RNAs [11±13]. Additionally, RNA modifications have recently been recognized as a prjsearch-list (grant number 31670824). Received new layer of epigenetic regulation, among which N6-methyladenosine (m6A) is the one being by B. -F. S. The funder had no role in study design, most extensively studied. m6A formation is catalyzed by a methyltransferase complex includ- data collection and analysis, decision to publish, or preparation of the manuscript. National Basic ing methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms' Research Program of China http://program.most. tumor 1-associating protein (WTAP), among which METTL3 functions as the catalytic sub- 6 gov.cn/ (grant number 2014CB964800). Received unit [14, 15]. As the most abundant and reversible modification on mRNAs, m A has been by W. L. The funder had no role in study design, proven to play key roles in regulating RNA stability [16] and RNA splicing [17], as well as data collection and analysis, decision to publish, or mRNA translation efficiency [18±22]. preparation of the manuscript. National Basic Many essential biological processes are known to be regulated by m6A, including cell fate Research Program of China https://www.360zhyx. 6 com/home-research-index-rid-66144.shtml (grant determination [23, 24] and embryonic development [24, 25, 26]. In neuronal systems, m A number 2016YFC0900300). Received by Y. -G. Y. has been shown to be a dynamic modification and increases in adulthood [27], suggesting a The funder had no role in study design, data potential function in neural plasticity and brain function. Previous studies have shown that collection and analysis, decision to publish, or conventional knockout of Mettl3 in mice leads to early embryonic lethality [24], and specific preparation of the manuscript. CAS Strategic depletion of m6A in the nervous system by conditional knockout of Mettl14 results in extended Priority Research Program http://www.cas.cn/kxyj/ index.shtml#xdzx (grant number XDB14030300). cell cycle and altered neurogenesis in embryonic mouse cortex, through regulating the decay 6 Received by Y. -G. Y. The funder had no role in of neurogenesis-related transcripts [28]. Furthermore, loss of m A eraser, the fat mass and study design, data collection and analysis, decision obesity-associated protein (FTO), leads to impaired adult neurogenesis and cognition ability to publish, or preparation of the manuscript. CAS [29]. FTO is enriched in axons, and specifically silencing axonal FTO inhibits axon elongation Key Research Projects of the Frontier Science by suppressing growth associated protein 43 (GAP-43) [30]. Nerve lesion induces the elevation http://www.cas.cn/jh/201701/t20170117_ of m6A level, and loss of METTL14 or m6A binding protein YTH N6-methyladenosine RNA 4588584.shtml (grant number QYZDY-SSW- SMC027). Received by Y. -G. Y. The funder had no binding protein 1 (YTHDF1) inhibits axonal regeneration [31]. All these studies indicate the 6 role in study design, data collection and analysis, important roles of m A signaling in neuronal development and neurogenesis. decision to publish, or preparation of