The 18S rRNA m 6 A methyltransferase METTL 5 promotes mouse embryonic stem cell differentiation

Article11 August 2020free access Source DataTransparent process The 18S rRNA m6A methyltransferase METTL5 promotes mouse embryonic stem cell differentiation Ming Xing State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Qi Liu State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, China Search for more papers by this author Cong Mao State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Hanyi Zeng State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Xin Zhang State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Shuqin Zhao State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Li Chen State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Mingxi Liu State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Bin Shen State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Xuejiang Guo State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Honghui Ma Corresponding Author [email protected] orcid.org/0000-0002-4167-1340 Key Laboratory of Arrhythmias of the Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Hao Chen Corresponding Author [email protected] orcid.org/0000-0002-2534-9650 School of Medicine, Southern University of Science and Technology, Shenzhen, China Search for more papers by this author Jun Zhang Corresponding Author [email protected] orcid.org/0000-0002-7630-8162 State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Ming Xing State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Qi Liu State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, China Search for more papers by this author Cong Mao State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Hanyi Zeng State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Xin Zhang State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Shuqin Zhao State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Li Chen State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Mingxi Liu State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Bin Shen State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Xuejiang Guo State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Honghui Ma Corresponding Author [email protected] orcid.org/0000-0002-4167-1340 Key Laboratory of Arrhythmias of the Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Hao Chen Corresponding Author [email protected] orcid.org/0000-0002-2534-9650 School of Medicine, Southern University of Science and Technology, Shenzhen, China Search for more papers by this author Jun Zhang Corresponding Author [email protected] orcid.org/0000-0002-7630-8162 State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China Search for more papers by this author Author Information Ming Xing1,‡, Qi Liu2,‡, Cong Mao1, Hanyi Zeng1, Xin Zhang1, Shuqin Zhao1, Li Chen1, Mingxi Liu1, Bin Shen1, Xuejiang Guo1, Honghui Ma *,3, Hao Chen *,4 and Jun Zhang *,1 1State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China 2State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, China 3Key Laboratory of Arrhythmias of the Ministry of Education of China, East Hospital, Tongji University School of Medicine, Shanghai, China 4School of Medicine, Southern University of Science and Technology, Shenzhen, China ‡These authors contributed equally to this work as first authors *Corresponding author. Tel: +86 021 61569704; E-mail: [email protected] *Corresponding author. Tel: +86 0755 88015592; E-mail: [email protected] *Corresponding author. Tel: +86 025 86869381; E-mail: [email protected] EMBO Rep (2020)21:e49863https://doi.org/10.15252/embr.201949863 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract RNA modifications represent a novel layer of regulation of gene expression. Functional experiments revealed that N6-methyladenosine (m6A) on messenger RNA (mRNA) plays critical roles in cell fate determination and development. m6A mark also resides in the decoding center of 18S ribosomal RNA (rRNA); however, the biological function of m6A on 18S rRNA is still poorly understood. Here, we report that methyltransferase-like 5 (METTL5) methylates 18S rRNA both in vivo and in vitro, which is consistent with previous reports. Deletion of Mettl5 causes a dramatic differentiation defect in mouse embryonic stem cells (mESCs). Mechanistically, the m6A deposited by METTL5 is involved in regulating the efficient translation of F-box and WD repeat domain-containing 7 (FBXW7), a key regulator of cell differentiation. Deficiency of METTL5 reduces FBXW7 levels and leads to the accumulation of its substrate c-MYC, thereby delaying the onset of mESC differentiation. Our study uncovers an important role of METTL5-mediated 18S m6A in mESC differentiation through translation regulation and provides new insight into the functional significance of rRNA m6A. Synopsis METTL5, the m6A methyltransferase for 18S rRNA, is critical for Fbxw7 translation and mESC differentiation. METTL5 is dispensable for mESC pluripotency. Orderly differentiation of mESCs requires METTL5. METTL5 promotes the translation efficiency of Fbxw7. Introduction To date, over 150 different types of chemical modifications have been identified in cellular RNAs, which brings out a new field termed as “epitranscriptome” (Helm & Motorin, 2017). Methylation of the N6 position of adenosine (m6A), as one of the most abundant mRNA modifications, also occurs on noncoding RNAs (ncRNAs) such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small nuclear RNAs (snRNAs) (Roundtree et al, 2017). The m6A modification on mRNAs has been extensively studied and demonstrated to regulate stability, splicing, nuclear export, and translation of mRNAs (Liu et al, 2015). Functional studies of m6A modification revealed that it is crucial for various physiological processes such as gametogenesis, neurogenesis, embryogenesis, and cell differentiation (Batista et al, 2014; Geula et al, 2015; Lin et al, 2017; Shi et al, 2018; Lan et al, 2019). rRNAs are the most abundant RNA species in cells and were reported to undergo a series of post-transcriptional modifications (Polikanov et al, 2015; Sloan et al, 2017). In general, eukaryotic rRNAs are highly decorated by 2′-O-methylation (Nm) and pseudouridine (Ψ) modifications, which are mostly introduced by box C/D and box H/ACA small nucleolar (sno)RNPs, respectively (Polikanov et al, 2015; Sloan et al, 2017). Previous study observed that loss of snoRNA-guided rRNA modification causes developmental defects and embryonic lethality in zebrafish (Higa-Nakamine et al, 2012). In addition, eukaryotic ribosomes contain several different types of base modifications, which are often installed by stand-alone rRNA-modifying enzymes (Sloan et al, 2017). Two site-specific m6A modifications were found at position 1,832 (m6A1,832) on 18S rRNA and position 4,220 (m6A4,220) on 28S rRNA in human cells and other vertebrates (Maden, 1986, 1988). Recently, ZCCHC4 was identified to be the human 28S rRNA m6A4,220 methyltransferase and is frequently overexpressed in tumors (Ma et al, 2019; Pinto et al, 2020). The depletion of ZCCHC4 significantly disrupts proper functions of translation in ribosomes, which in turn affects cell proliferation and cancer progression. Thus, rRNA modifications are critical in regulating ribosome function and intricately linked to development and tumorigenesis. Mouse embryonic stem cells (mESCs) are derived from the inner cell mass (ICM) of late pre-implantation blastocysts and have the capability to differentiate into a variety of cell types comprising the derivatives of three germ layers (Evans & Kaufman, 1981). The in vitro differentiation system has been used extensively to study stem cell pluripotency and cell fate decisions (Pedersen, 1994; Keller, 1995). Multiple regulatory mechanisms involving transcriptome, epitranscriptome, translatome, and proteome precisely orchestrate cell fate determination (Szutorisz et al, 2006; Lu et al, 2009; Meissner, 2010; Kojima et al, 2014; Shi et al, 2017). Among them, ubiquitin–proteasome system (UPS) emerges as an essential regulator of stem cell function (Buckley et al, 2012; Strikoudis et al, 2014). For example, F-box and WD-40 domain 7 (FBXW7), a recognition component of SCF (complex of SKP1, CUL1 and F-box protein)-type ubiquitin ligase, was identified as an important factor for ESC differentiation and cellular reprogramming in UPS-targeted RNAi screens (Buckley et al, 2012). Following-up studies showed that silencing of FBXW7 expression inhibits ESC differentiation by stabilization of its key substrate c-MYC, which positively regulates pluripotency-related networks to maintain ESC self-renewal and hinders differentiation (Smith & Dalton, 2010; Fagnocchi et al, 2016). However, how Fbxw7 itself is regulated by upstream factors during these biological processes is still open to question. Recently, several methyltransferase-like (METTL) family proteins have been reported to play vital roles in regulating ESC pluripotency and early embryonic development. For instance, METTL3-METTL14 methyltransferase complex ensures pluripotent cell differentiation, which is dependent on its m6A catalytic activity (Batista et al, 2014; Geula et al, 2015). mRNA m3C writer METTL8 inhibits c-Jun N-terminal kinase (JNK) pathway and affects mESC differentiation (Xu et al, 2017; Gu et al, 2018). U6 snRNA m6A methyltransferase METTL16 is required for mouse embryonic development via controlling the splicing of SAM synthetase MAT2A (Pendleton et al, 2017; Mendel et al, 2018). Though a previous study firstly analyzed the atomic-resolution structure of METTL5 and identified the 18S rRNA m6A role of METTL5 in human cancer cells during the preparation of our work (van Tran et al, 2019), the exact roles of 18S rRNA m6A on translation regulation are still largely unknown. Here, we showed that METTL5 is responsible for 18S rRNA methylation both in vivo and in vitro. Functionally, m6A-dependent METTL5 endows mESCs with efficient translation for triggering multi-lineage differentiation timely, which depicts the importance of rRNA m6A in cell fate transition. Results METTL5 is the N6-adenosine methyltransferase targeting 18S rRNA METTL5, as a METTL family member, has been reported to bind RNA rather than DNA (Franke et al, 2015). Through bioinformatic analysis, we identified that there is a typical [DNSH] PP [YFW] motif of m6A-specific methyltransferase in METTL5 protein (Fig EV1A), which inspired us to search for its potential targets and biological function. Previous functional studies uncovered the crucial roles of METTL family members in regulating pluripotency of mESCs (Batista et al, 2014; Geula et al, 2015; Gu et al, 2018). We were thus prompted to use the mESC model to determine the function of METTL5. Click here to expand this figure. Figure EV1. METTL5 is a putative N6-adenosine methyltransferase Schematic representation of the functional domain of mouse METTL5. The NPPF motif was highlighted with a red box. APPA motif was the catalytically inactive mutant after changing key residues (126–129) “NPPF” to “APPA”. PAGE gel electrophoresis showing the PCR amplification of the targeted region of Mettl5 locus from WT and Mettl5 KO mESCs. Sequencing results of the targeted region Mettl5 locus detected in WT and KO clones in mESCs. Red colored letters indicate Mettl5 sgRNA sequence. Green colored and underlined letters indicate Mettl5 PAM sequence. Blue colored dots and letters indicate deletions and insertions, respectively. 15 TA clones of the PCR products were analyzed by Sanger sequence. LC/MS analysis of the m6A level of total RNA from the WT and Mettl5 KO (KO-6B) mESCs. Data are shown as mean ± SD from three biological replicates. Student's t test, two-tailed. ***P < 0.001. Real-time fluorescence amplification curves of qPCR showing the SELECT results for detecting m6A1,832 and A1,825 sites (for input control). Histogram showing the threshold cycles of qPCR (qPCRCT) for detecting m6A1,832 and A1,825 sites in 18S rRNA of WT and Mettl5 KO (KO-6B) mESCs, respectively. Data are shown as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; ***P < 0.001. Coomassie blue staining showing the purified WT and catalytically mutant METTL5 proteins. Source data are available online for this figure. Download figure Download PowerPoint First, CRISPR/Cas9 system was used to target the second exon of Mettl5 to generate two Mettl5 knockout (KO) colonies (number KO-2C and KO-6B; Fig 1A). The successful depletion of Mettl5 was validated both by Sanger sequencing and Western blot (WB) using METTL5-specific antibody (Figs 1B and EV1B and C). Consistent with our hypothesis that METTL5 may function as a m6A methyltransferase, we observed a dramatic decrease of m6A level in total RNA from Mettl5 KO cells compared with that in wild-type (WT) cells (Fig EV1D). Considering the rRNA is the most abundant RNA type in the cell, we wondered whether the loss of m6A is actually from rRNA. To test this hypothesis, we quantified the m6A levels of purified 18S and 28S rRNA from WT and KO cells with LC-MS/MS (QQQ). As expected, a dramatic decrease of m6A was detected in 18S rRNA from Mettl5 KO cells. In line with recent reports that ZCCHC4 is a specific 28S rRNA m6A methyltransferase (Ma et al, 2019; Pinto et al, 2020), we did not detect any noticeable changes of m6A level in 28S rRNA (Fig 1C). Furthermore, we employed SELECT method with the accuracy of single-base resolution to confirm 18S rRNA m6A at position 1,832 indeed installed by METTL5 (Xiao et al, 2018). Consistently, the SELECT assay data indicated that m6A1,832 level on 18S rRNA was significantly decreased upon Mettl5 KO, enabling the ligation products more sufficient for amplification (Figs 1D and EV1E and F). Then, we set out to investigate whether METTL5 can install a methyl group into the 18S rRNA in vitro. We purified the WT and catalytically inactive mutant (“NPPF” to “APPA”, Mut) recombinant METTL5 proteins from E. coli and then incubated them with synthetic 18S rRNA oligos, respectively (Fig EV1G). The in vitro assay results showed that WT METTL5 proteins have considerable methyltransferase activity toward 18S rRNA oligos. In sharp contrast, there was barely any activity detected in Mut METTL5 group (Fig 1E), indicating that METTL5 is a bona fide m6A methyltransferase for 18S rRNA in vivo and in vitro, which is similar with the recent report (van Tran et al, 2019). Figure 1. METTL5 is the N6-adenosine methyltransferase targeting 18S rRNA in vivo and in vitro Schematic diagram of sgRNA targeting mouse Mettl5 locus. sgRNA targeting site is highlighted in red. PAM sequence is underlined and highlighted in green. Western blot analysis confirming two Mettl5 KO clones by specific METTL5 antibody. α-TUBULIN was used as a loading control. LC-MS/MS analysis of m6A/A levels in purified 18S rRNA and 28S rRNA from the WT and Mettl5 KO (KO-6B) mESCs. PAGE gel electrophoresis showing the PCR amplification of the elongated and ligated products of SELECT method for detecting m6A1,832 site and A1,825 site (for input control) in 18S rRNA of WT and Mettl5 KO (KO-6B) mESCs, respectively. LC-MS/MS analysis of d3-m6A/A levels in 18S rRNA oligos after being incubated with recombinant WT or catalytically mutant mouse METTL5 proteins in vitro. No enzyme group was used as a negative control. Source data are available online for this figure. Source Data for Figue 1 [embr201949863-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Mettl5 KO mESCs exhibit unaffected pluripotency and self-renewal Upon deleting Mettl5, we firstly noticed that mESCs still maintained nest, round colony morphologies (Fig 2A). Alkaline phosphatase (AP) staining showed that Mettl5 KO mESCs had clearly positive reactivities similar to those of WT mESCs when cultured in the naive 2i/Lif condition (Fig 2B). Through detecting the mRNA and protein levels of pluripotency markers such as POU5F1, SOX2, NANOG, and ESRRB by real-time quantitative PCR (RT–qPCR) and WB, respectively, we found that expressions of pluripotency-related factors were almost unaltered in Mettl5 KO mESCs (Fig 2C and D). Moreover, immunostaining analysis of POU5F1, NANOG, and SOX2 in mESCs indicated that depletion of Mettl5 does not disrupt the undifferentiated status of mESCs (Fig 2E). Additionally, Mettl5 KO mESCs also displayed similar pluripotency as WT mESCs when cultured in the conventional serum/Lif condition (Fig EV2A–D). Hence, these results indicated that METTL5 is dispensable for maintaining pluripotency and self-renewal of mESCs. Figure 2. Characterizations of the Mettl5 KO mESCs A, B. Representative bright-filed (A) and AP staining (B) images of the WT and Mettl5 KO mESCs cultured in 2i/Lif medium. Scale bars represent 100 and 50 μm, respectively. C. RT–qPCR analysis of the expression of pluripotency genes in WT and Mettl5 KO mESCs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; ***P < 0.001. D. Western blot analysis of the expression of pluripotency markers in WT and Mettl5 KO mESCs. α-TUBULIN was used as a loading control. E. Immunostaining analysis of the expression of pluripotency markers (POU5F1, SOX2 and NANOG) in WT and Mettl5 KO mESCs, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 10 μm. Source data are available online for this figure. Source Data for Figue 2 [embr201949863-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Characterizations of the Mettl5 KO mESCs in serum/Lif condition A, B. Representative bright-filed (A) and AP staining (B) images of WT and Mettl5 KO mESCs. Scale bars represent 100 and 50 μm, respectively. C. RT–qPCR analysis of the expression of pluripotency genes. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; ***P < 0.001. D. Western blot analysis showing the expression of pluripotency markers in WT and Mettl5 KO mESCs. α-TUBULIN was used as a loading control. Source data are available online for this figure. Download figure Download PowerPoint Depletion of Mettl5 impairs mESC early differentiation We next sought to explore the role of METTL5 in differentiation potential using a three-dimensional (3D) model of inducing embryoid body (EB) formation. Due to the greater differentiation capacity than serum/Lif-grown mESCs, 2i/Lif-grown mESCs were chosen to induce differentiation (Marks et al, 2012). The mESCs can be spontaneously differentiated into multi-lineage cells under the defined serum-free suspension culture (SFEB) condition after withdrawal of all cytokines (Fig EV3A) (Pedersen, 1994; Keller, 1995). As expected, POU5F1 expression gradually decreased during the course of differentiation, indicating that the cell aggregates were switching off pluripotency gene regulatory networks. Intriguingly, the endogenous METTL5 protein expression increased to its peak at the early differentiation stage of Day3 and then recovered to a previous level (Fig 3A). Consistently, the m6A1,832 modification level was transiently increased in Day3 EBs, compared with that in other time points detected by SELECT (Figs 3B and EV3B and C), which implied that METTL5 might play a certain role in initiating early differentiation. Click here to expand this figure. Figure EV3. Induction of embryoid body formation in vitro Representative bright-filed images of EBs at different time points. Scale bars represent 20 μm. Real-time fluorescence amplification curves of qPCR showing the SELECT results for detecting m6A1,832 and A1,825 sites (for input control) during the indicated time points of EB induction. Histogram showing the threshold cycles of qPCR (qPCRCT) for detecting A1,825 site in 18S rRNA during the indicated time points of EB induction. Data are shown as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant. Immunostaining analysis of the mesoderm marker T in control and Mettl5 KO EBs at different time points. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 100 μm. Quantification of percentages of positive cells shown in (D). Quantitative analysis was based on at least three independent experiments. Data are presented as mean ± SD. Student's t test, two-tailed. **P < 0.01; ***P < 0.001. Representative AP staining images of the control and Mettl5 KO Day7 EBs. Scale bars represent 20 μm. Western blot analysis confirming overexpression of HA-tagged WT or catalytically mutant (aa126–129: “NPPF” to “APPA”) METTL5 in KO mESCs, respectively. α-TUBULIN was used as a loading control. Western blot analysis showing the expression of pluripotency (POU5F1) and three germ layer markers (FOXA2, T and NESTIN) in Day7 KO, KO + HA-MT5WT, and KO + HA-MT5Mut EBs. GAPDH was used as a loading control. H&E staining and immunostaining analysis revealing the differentiated structures of three germ layers in teratomas derived from WT and Mettl5 KO mESCs. The mESCs were injected subcutaneously into the flanks of immunodeficient mice (n = 6 per group). Scale bars represent 50 μm (H&E staining) and 20 μm (immunostaining), respectively. Source data are available online for this figure. Download figure Download PowerPoint Figure 3. Mettl5 KO impairs early differentiation of mESCs Western blot analysis showing dynamic expression patterns of POU5F1 and METTL5 during the indicated time points of EB induction. α-TUBULIN was used as a loading control. Histogram showing the threshold cycles of qPCR (qPCRCT) for detecting m6A1,832 site in 18S rRNA during the indicated time points of EB induction. Data are shown as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; *P < 0.05. Immunostaining analysis of the expression of pluripotency marker POU5F1(left), endoderm marker FOXA2 (middle), and neuroectoderm marker NESTIN (right) in control and Mettl5 KO EBs at different time points, respectively. DAPI (blue) was used as a nuclear counterstain. Scale bars represent 100 μm. Quantification of the percentages of positive cells shown in (C). Quantitative analysis was based on at least three independent experiments. Data are represented as mean ± SD. Student's t test, two-tailed. **P < 0.01; ***P < 0.001. RT–qPCR analysis of the expressions of pluripotency and lineage-specific markers in Day7 EBs. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Western blot analysis showing pluripotency (POU5F1) and three germ layer markers (FOXA2, T and NESTIN) expression in Day7 EBs. α-TUBULIN was used as a loading control. Source data are available online for this figure. Source Data for Figue 3 [embr201949863-sup-0006-SDataFig3.pdf] Download figure Download PowerPoint To investigate the potential role of METTL5 during the differentiation, we quantified the expressions of pluripotency and lineage-specific markers in EBs by immunostaining analysis at the indicated time points of differentiation. Encouragingly, we found that expression levels of pluripotency marker POU5F1 were strikingly higher than the controls after Day3, and lineage-specific markers such as FOXA2 (for endoderm), T (for mesoderm), and NESTIN (for neuroectoderm) failed to be timely expressed in Mettl5 KO EBs (Figs 3C and D, and EV3D and E), suggesting that Mettl5 KO mESCs were resistant to the stimuli of triggering differentiation. Then, Day7 EBs were harvested for more detailed analysis. AP staining showed the control EBs were undergoing differentiation and maturation, whereas Mettl5 KO EBs still remained high pluripotency with dark purple staining (Fig EV3F). Both of the RT–qPCR and WB analysis confirmed that the naive pluripotency markers (POU5F1, NANOG, and ESRRB) were still highly expressed, while the endoderm markers (FOXA2, SOX17, GATA4, and GATA6) and mesoderm markers (MIXL1, WNT3, and T) were severely suppressed in Mettl5 KO EBs, compared with the controls (Fig 3E and F). To determine whether the deficient differentiation was indeed caused by the loss of 18S rRNA m6A, we performed a rescue experiment by overexpressing either HA-tagged WT or catalytically mutant (“NPPF” to “APPA”, Mut) METTL5 in KO cells (KO + HA-MT5WT or KO + HA-MT5Mut; Fig EV3G). As shown in Fig EV3H, differentiation block was rescued by re-expressing HA-tagged WT METLL5 rather than Mut METTL5, demonstrating that the regulation is dependent on the m6A catalytic activity of METTL5. Nevertheless, we analyzed the 6-week teratomas derived from WT and Mettl5 KO mESCs. Histological analysis of sections revealed that both groups were composed of characteristic lineage-specific morphologies and markers such as epithelia (for endoderm), muscle (for mesoderm), and epidermis (for ectoderm) (Fig EV3I). Therefore, Mettl5 KO does not fully inhibit differentiation potential in vivo and mESCs can ultimately differentiate into three germ layers. Collectively, these data suggested that disruption of m6A-dependent METTL5 delays pluripotency exit and impairs germ layer specification of mESCs. Depletion of Mettl5 affects translation efficiency of mRNAs Considering that 18S A1,832 is localized in the decoding center (DC) (Piekna-Przybylska et al, 2008), we thus focused on the role of METTL5 in the translation regulation. Ribosome profiling sequencing (Ribo-seq) was performed to chart the translational profiles in WT and Mettl5 KO mESCs. The initial quality control showed that a majority of footprints mapped to coding sequences (CDS) with the strong 3-nt periodicity distribution (Fig EV4A–C). Thus, the data quality was good enough for downstream analysis (Calviello & Ohler, 2017). The translation efficiency correlation analysis showed there was a good consistency between two replicates in both WT and Mettl5 KO cells (Fig EV4D and E). Principal component analysis (PCA) revealed that two replicates from WT and KO groups were clustered, respectively, whereas the two groups were separated clearly from each other, reflecting a large divergence between the WT and Mettl5 KO cells (Fig EV4F). We identified 1,339 differentially translated genes (1,169 down-regulated and 170 up-regulated genes) between WT and KO cells in a total of 9,093 actively transcribed genes with high confidence (≥ 1.5-fold change and P < 0.05) (Fig 4A and Dataset EV1). Notably, core stemness genes such as Pou5f1, Nanog, Sox2, and Esrrb exhibited non-significantly translational changes in Mettl5 KO mESCs compared with controls, in agreement with the findings as described in Fig 2. Click here to expand this figure. Figure EV4. Quality control of Ribo-seq data in mESCs A. The