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Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase

Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase Cell Research (2014) 24:177-189. npg © 2014 IBCB, SIBS, CAS All rights reserved 1001-0602/14 www.nature.com/cr ORIGINAL ARTICLE Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase 1, 8, * 1, * 2, * 1, 8, * 1 1 1 Xiao-Li Ping , Bao-Fa Sun , Lu Wang , Wen Xiao , Xin Yang , Wen-Jia Wang , Samir Adhikari , 1 1 1 1 1 1 1 3 4 Yue Shi , Ying Lv , Yu-Sheng Chen , Xu Zhao , Ang Li , Ying Yang , Ujwal Dahal , Xiao-Min Lou , Xi Liu , 5 6 6 6 1 4 Jun Huang , Wei-Ping Yuan , Xiao-Fan Zhu , Tao Cheng , Yong-Liang Zhao , Xinquan Wang , 1, 7 2 1, 8 Jannie M Rendtlew Danielsen , Feng Liu , Yun-Gui Yang Center For Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China; State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Chinese Academy of Sciences Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China; Center for Structural Biology, Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China; Life Sciences Institute, Zhejiang University, Zhejiang 310058, China; State Key Laboratory of Experimental Hematology, Institute of Hematology, Tian- jin 300041, China; The Novo Nordisk Foundation Center for Protein Research, Ubiquitin Signalling Group, Faculty of Health Sciences, Copenhagen, Denmark; University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms’ tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic ac- tivity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-en- hanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransfer - ase complex and play a critical role in epitranscriptomic regulation of RNA metabolism. Keywords: WTAP; m6A methyltransferase; METTL3; METTL14; mRNA Cell Research (2014) 24:177-189. doi:10.1038/cr.2014.3; published online 10 January 2014 Adomet) serving as the methyl donor [1-3]. The N6- Introduction methyladenosine (m6A) modification is one of the most Nitrogen or oxygen atoms in RNA are ubiqui - frequent methylations in eukaryotic mRNA, accounting tously methylated with S-adenosylmethionine (SAM, for over 80% of all RNA base methylations and, it has been observed in various species [4-8]. Recently, two independent studies combining m6A immunoprecipita- *These four authors contributed equally to this work. tion with high-throughput analysis revealed that the m6A a b Correspondence: Yun-Gui Yang , Feng Liu modification tends to mainly occur in intragenic regions. E-mail: [email protected] The frequency is particularly high in the 3′-end of cod- E-mail: [email protected] ing sequence (CDS) and the first quarter of the 3′-UTR, Received 13 October 2013; revised 3 December 2013; accepted 4 Decem- ber 2013; published online 10 January 2014 with an average of 1 m6A per 2 000 ribonucleotides. The npg WTAP as m6A methyltransferase regulatory subunit m6A modification occurs in highly conserved regions 28]. METTL14 protein shares 43% identity (Supple- with the consensus sequence, RRACH (R = G or A; H = mentary information, Figure S1B) with METTL3, and A, C or U) [9-13]. During brain development a dynamic a previous phylogenetic analysis has suggested that the change in m6A levels on RNA has been observed [10], two proteins are homologs [23]. Furthermore, METTL14 suggesting the existence of RNA m6A demethylase(s). contains similar motifs essential for catalytic activity as Indeed, two AlkB dioxygenase family proteins, fat mass METTL3 (Supplementary information, Figure S1B). The and obesity associated gene (FTO) and ALKBH5, were interactions among METTL3, WTAP and METT14 were recently demonstrated to catalyze the removal of the confirmed by co-immunoprecipitation (Figure 1A, 1E m6A mark both in vitro and in vivo [14, 15]. These two and Supplementary information, Figure S2A and S2B). enzymes function in various biological processes such The specificity of the interaction between METTL3 and as development, metabolism, and fertility. They regulate WTAP was verified by examination of the endogenous the expression levels of thousands of genes, suggesting a interaction between these two proteins after depletion pivotal epitranscriptomic function of m6A modification of WTAP by small interfering RNAs (siRNA) (Figure in regulating RNA metabolism [8, 16-19]. 1B). GST pull-down using purified recombinant GST- In contrast to the discovery of RNA m6A demethyl- METTL3 and 6×His-WTAP proteins demonstrated a di- ases, the nature of the methyltransferase complex respon- rect interaction between METTL3 and WTAP (Figure 1C sible for catalyzing m6A formation in RNA still remains and Supplementary information, Figure S1C). By using largely unknown. The mammalian m6A methyltransfer- WTAP constructs with either the C- (WTAP-N, 1-200 ase complex is generally perceived to contain at least two aa) or N-terminus (WTAP-C, 201-396 aa) truncated, we key subcomplexes designated MT-A (200 kD) and MT-B found that the interaction between WTAP and METTL3 (800 kD) [20, 21]. So far only a 70-kD protein, methyl- or METTL14 depends on the N-terminus of WTAP (Fig- transferase like 3 (METTL3) has been identified as one ure 1D and 1F). It has been reported that RNA is not component of the 200-kD MT-A complex. METTL3 ho- an essential component of the m6A methyltransferase mologs have been identified in S. cerevisiae, Drosophila complex [20, 29]. Consistently, we observed that nei- and Arabidopsis [22-26]. Although mRNA methylation ther RNase A treatment nor inhibition of methylation by occurs primarily within the RRACH consensus sequence cycleoleucine affected the interaction between WTAP in mammals, only a portion of the RRACH sites contain and METTL3 (Supplementary information, Figure S2C- the m6A modification [9, 10, 27]. Mechanistic under- S2E). Based on these findings, we conclude that WTAP standing of the selectivity, regulation and function of the and METTL14 are bona fide components of the m6A m6A modification depends on a complete characteriza- methyltransferase complex, and that both RNA and the tion of the methyltransferase complex. m6A modification are dispensable for the interaction In this study, we identify for the first time, Wilms’ between WTAP and METTL3. In the remaining of this tumor 1 (WT1)-associating protein (WTAP) and meth- paper, we will refer to this complex as the WMM (WTAP, yltransferase like 14 (METTL14) as components of the METTL3 and METTL14) complex. mammalian m6A methyltransferase complex. In particu- lar, WTAP appears to serve as a regulatory subunit essen- WTAP is required for m6A methyltransferase activity in tial for m6A methyltransferase activity. vivo WTAP does not harbor any obvious catalytic domains, and in contrast to METTL3 and METTL14 that were Results recently demonstrated to form an active complex capable Identification of WTAP and METTL14 as METTL3-asso - of catalyzing m6A formation, WTAP showed no catalytic activity itself or effect on the activity of METTL3-MET- ciating proteins TL14 complex in vitro [30]. Given the direct interaction To identify mammalian METTL3-associating pro- between WTAP and METTL3, we speculated that WTAP teins, we utilized tandem affinity purification to pull may serve to regulate m6A methyltransferase activity down stably expressed SFB-tagged METTL3 from 293T in vivo. To test this hypothesis, we examined the m6A cells. Subsequent mass spectrometry analysis of affinity- levels in mRNA extracted from WTAP- or METTL3- purified SFB-METTL3 protein complexes identified knockdown cells. Dot-blot assays showed that depletion two potential METTL3-associating proteins, WTAP and of either of these proteins resulted in significantly lower METTL14 (Supplementary information, Figure S1A). m6A levels in mRNA (Figure 2A and Supplementary in- Consistently, AtFIP37 and Mum2, the WTAP homologs formation, Figure S3A). RT-PCR and western blot analy- in plant and yeast, respectively, have previously been ses verified the knockdown efficiency and specificity, shown to associate with METTL3 in these species [26, Cell Research | Vol 24 No 2 | February 2014 npg Xiao-Li Ping et al . Figure 1 WTAP interacts with METTL3 and METTL14. (A) 293T cells were transfected with Flag-WTAP and Myc-METTL3 constructs as indicated. Forty-eight hours later, cells were lysed and the lysates were subjected to immunoprecipitation using anti-Myc (Myc-IP) followed by immunoblotting with the anti-Flag antibodies. (B) 293T cells were treated with control siRNA (siCTRL) or siRNA targeting WTAP (siWTAP) for 48 h. Then cells were lysed and the lysates were subjected to IP using anti- WTAP. The immunoprecipitated samples were analyzed by immunoblotting with the anti-METTL3 antibodies. (C) Purified recombinant His-WTAP proteins were mixed with either GST or GST-METTL3 proteins as indicated, pulled down with GST beads, and subjected to immunoblotting with the indicated antibodies. (D) 293T cells were co-transfected with Myc-METTL3 and Flag-WTAP full-length (-FL), N-terminal (-N) or C-terminal (-C) constructs as indicated. Forty-eight hours later, cells were lysed and the lysates were subjected to Myc-IP followed by immunoblotting with the anti-Flag antibodies. (E) 293T cells were transfected with Flag-WTAP and HA-METTL14 constructs as indicated. Forty-eight hours later cells were lysed and the ly- sates were subjected to HA-IP followed by immunoblotting with the anti-Flag antibodies. (F) 293T cells were co-transfected with HA-METTL14 and Flag-WTAP full-length (-FL), N-terminal (-N) or C-terminal (-C) constructs as indicated. Forty-eight hours later, cells were lysed and the lysates were subjected to HA-IP followed by immunoblotting with the anti-Flag antibod- ies. Supportive data were included in Supplementary information, Figures S1 and S2. www.cell-research.com | Cell Research npg WTAP as m6A methyltransferase regulatory subunit respectively (Supplementary information, Figure S3B role in regulating m6A modification is independent of and S3C). Moreover, depletion of WTAP or METTL3 its previously described binding partner WT1 (Figure did not affect the expression of the other complex com- 2A and Supplementary information, Figure S3A). Taken ponents (Supplementary information, Figure S3B and together, these findings suggest that WTAP affects m6A S3C). WTAP was originally identified as a protein asso - methyltransferase activity in vivo. ciated with WT1 [31]. We examined whether WT1 might also be required for m6A methyltransferase activity in WTAP r egulates accumulation of METTL3 and vivo. Interestingly, we found that WT1 depletion had no METTL14 in nuclear speckles influence on m6A abundance, suggesting that WTAP’s Previous studies have indicated that m6A modification Figure 2 WTAP regulates the nuclear speckle localization of METTL3 and METTL14. (A) The graph represents the quantifi - cation of three independent dot-blot experiments (raw data were included in Supplementary information, Figure S3A). The y- axis represents the relative intensity of dots relative to that of the control group. P values were calculated using a two-tailed t- test. Error bars represent SD. (B-D) After transfection (48 h) with the indicated fluorescence FAM-labeled siRNA, HeLa cells were fixed and immunostained with the indicated antibodies. DNA was visualized by DAPI. Scale bar, 10 μm. Supportive data were included in Supplementary information, Figure S3. Cell Research | Vol 24 No 2 | February 2014 npg Xiao-Li Ping et al . occurs at the pre-mRNA stage within the nucleus and it and fixation procedures. A similar staining pattern in nuclear speckles has also previously been reported [15]. thereby may affect splicing and mRNA export [2, 32- 35]. Nuclear speckles are enriched in proteins involved mRNA is the major RNA species bound by WTAP and in RNA processing and alternative splicing. Interest- ingly, METTL3 and WTAP as well as the m6A demeth- METTL3 We next performed Photoactivatable-Ribonucleoside- ylases FTO and ALKBH5 have previously been shown to colocalize with nuclear speckle markers [14, 15, 31]. Enhanced Crosslinking and Immunoprecipitation (PAR- CLIP) [51] to identify the targets of WTAP and METTL3 The physical interaction between WTAP and METTL3 and METTL14 prompted us to examine the cellular proteins. In brief, 4-thiouridine (4-SU), a photoreactive ribonucleoside analog, was incorportated into RNA tran- distribution of METLL14. Using immunofluorescence microscopy, we found that endogenous METTL14, like scripts, which leads to the thymidine-to-cytidine muta- tion in crosslinked sequences. Therefore, one unique METTL3 and WTAP, exhibits a diffused nucleoplasmic staining pattern with intense granule-like foci, and that feature of cDNA libraries prepared by PAR-CLIP is that the precise position of crosslinking can be identified by it colocalizes well with the pre-mRNA splicing factor SC35 in nuclear speckles (Figure 2C and Supplementary mutations residing in the sequenced cDNA. PARalyzer [36, 37] software was used to identify the preferred re- information, Figure S3F). Interestingly, WTAP deple- tion decreased the accumulation of both METTL3 and gions bound by WTAP and METTL3. 4 986 METTL3- and 922 WTAP-binding clusters were enriched and could METTL14 in nuclear speckles (Figure 2B, 2C and Sup- plementary information, Figure S3G), whereas knock- be assigned into four RNA categories: mRNA, miRNA, large intergenic non-coding RNA (lincRNA) and pseu- down of either METTL3 or METTL14 had no effect on the localization of WTAP (Figure 2D and Supplementary dogene (Figure 3A). About 95% and 92% of the RNA bound by METTL3 and WTAP, respectively, represented information, Figure S3G). Furthermore, METTL3 deple- tion affected the nuclear speckle localization of MET- mRNA, demonstrating that mRNA is the major substrate of the WMM complex. We further mapped the mRNA TL14 and vice versa (Figure 2B and 2C). Consistent with our observations on m6A methyltransferase activity, clusters into four non-overlapping transcript regions, 5′- UTR, CDS, 3′-UTR and intron, according to the human WT1 depletion did not affect METTL3 or WTAP local- ization (Figure 2B and 2D). Based on these observations, reference transcript annotation. After normalization of the number of clusters to the overall length of the dif- we conclude that WTAP is required for accumulation of METTL3 and METTL14 in nuclear speckles. ferent transcript regions, the majority of binding sites of WTAP and METTL3 were identified in CDS and UTR Nuclear speckle-associating factors can be classified as RNase-sensitive or -insensitive factors based on the regions (Figure 3B). To define the in vivo RNA recognition elements for nature of the association with the nuclear speckles. To characterize the nature of the association of the WMM WTAP and METTL3, the binding cluster data were ana- lyzed by HOMER, a suite of tools for motif discovery complex with nuclear speckles, we treated cells with RNase A. Similar to the mRNA processing factor SM and next-generation sequencing analysis [38]. In this analysis procedure, the WTAP and METTL3 clusters (smith antigen), but unlike the splicing factor SC35, RNase A treatment resulted in a dramatic decrease of were set as the target sequences, and a set of background clusters was generated with the BEDTools’ shuffleBed WMM complex accumulation in nuclear speckles, dem- onstrating that the WMM complex associates with RNA, program [39] to randomly shuffle regions of the same size as the clusters throughout the gene regions, with which is essential for the recruitment and/or retention of the WMM complex in nuclear speckles (Supplementary the parameter for HOMER motif length from 5 to 8. The motifs AGGACU (P = 1e-14) and UGUGGACU information, Figure S3D-S3F). It is worth noting that METTL3 and METTL14 staining almost disappeared (P = 1e-13) were enriched in WTAP- and METTL3- binding clusters, respectively (Figure 3C). When we only upon WTAP knockdown, but the overall protein levels of METTL3 and METTL14 did not change following included genes found in both WTAP- and METTL3- binding clusters, the highest scoring motif was UUAG- WTAP depletion (Supplementary information, Figure S3B and S3C). It is likely that when WTAP is depleted, GACU (P = 1e-19) (Figure 3C). In addition, AGGAC METTL3 and METTL14 no longer bind to RNA in the (P = 1e-12), UGGAC (P = 1e-12), and AGACUAA (P nuclear speckles, and become diffused within the entire = 1e-10) were also highly enriched in WTAP, METTL3 cell, which reduces the staining density to levels below and WTAP/METTL3 overlay clusters, respectively (Fig- detection. In addition, unbound METTL3 and METTL14 ure 3D and Supplementary information, Figure S4B). might easily get washed out during the permabilization This is in accordance with the reported consensus m6A www.cell-research.com | Cell Research npg WTAP as m6A methyltransferase regulatory subunit Cell Research | Vol 24 No 2 | February 2014 npg Xiao-Li Ping et al . motif RRACH (R = G or A; H = A, C or U) [9, 10]. The Taken together, these findings support the existence of high degree of similarity of the mRNA binding motifs a WTAP-METTL3-containing complex that methylates between WTAP and METTL3 are consistent with the re- RNA at specific sites in mammals. sults that WTAP and METTL3 form a complex. We next calculated the distance between WTAP/ The WMM complex regulates transcription and METTL3 clusters and the m6A sites. To obtain the best alternative splicing coverage of m6A sites, we downloaded human m6A data Gene Ontology (GO) analyses of mRNA captured by released by Dominissini et al. [9] and Meyer et al. [10] METTL3 and WTAP using PAR-CLIP revealed that a and aligned the data to the hg19 genome by Tophat2 [40]. large proportion of mRNA species associated with both We then used MACS [41] and Fisher test [9, 10] meth- proteins were derived from genes involved in transcrip- ods to determine m6A peaks. The m6A peaks identified tion and RNA metabolism/splicing (Supplementary in this analysis were combined with the previously pub- information, Figure S5A). To further investigate the po- lished m6A sites, and the duplicates were removed. Then tential roles of the WMM complex in RNA metabolism, we used the BEDTools’ closestBed [39] to calculate the transcriptome analyses were performed to compare the distance between WTAP/METTL3 clusters and m6A genome-wide gene expression and alternative splicing sites. We generated control clusters with BEDTools’ events between wild-type cells and WTAP- or METTL3- shuffleBed [39] to randomly shuffle regions of the same deficient cells. The transcriptome analyses revealed a sig- nificant overlap in differentially expressed genes (DEGs) size as the clusters. The result demonstrated a significant spatial correlation (P = 2.2e-16) with 74% WTAP- and between WTAP- and METTL3-deficient cells (Supple- mentary information, Figure S5C). Consistent with the 69% METTL3-binding clusters in CDS and UTR regions overlapping with m6A sites (Figure 3E and Supplemen- GO analysis of the PAR-CLIP data, GO analysis of the overlapping DEGs from WTAP- and METTL3-deficient tary information, Figure S4C and S4D). These results strongly suggest a significant role of both WTAP and cells demonstrated that most of these genes were related to transcription and RNA processing (Supplementary METTL3 in m6A modification. The results that WTAP is required for nuclear speckle information, Figure S5D), further emphasizing the poten- tial significance of WTAP and METTL3 in RNA metabo - localization of METTL3 and METTL14 and for m6A modification prompted us to test whether the absence of lism. Interestingly, both METTL3 and WTAP knockdowns WTAP might influence the binding of METTL3 to RNA. Interestingly, we found a significantly reduced amount resulted in a pronounced variation in isoform numbers with about half of the variations (2 296) present in both of RNA associated with METTL3 upon WTAP depletion (Figure 3F and Supplementary information, Figure S5B), WTAP- and METTL3-deficient cells (Supplementary information, Figure S5E). When comparing the 2 296 suggesting that WTAP is likely responsible for recruit- ing the m6A methyltransferase complex to target RNAs. genes with changes in isoform numbers with the 410 Figure 3 METTL3 and WTAP bind m6A consensus sequence in mRNA and affect gene expression and alternative splicing. (A) Percentage of various RNA species bound by WTAP and METTL3 based on PAR-CLIP analyses. The WTAP- and METTL3- binding clusters were identified by PARalyzer algorithm. Annotation of clusters was based on the human Ensembl gtf file (ver- sion 72, hg19). The majority of binding sites of WTAP and METTL3 were located in mRNA. (B) Pie chart depicting the distri- bution of binding clusters in mRNA based on PAR-CLIP sequence clusters for WTAP and METTL3 after normalization of the overall length of the different transcript regions. Length of these different transcript regions was extracted from Ensembl an- notations and the distribution percentage of clusters in these regions were normalized by their length. (C) Enriched sequence motif analysis of binding clusters indentified by PAR-CLIP. Upper panel, WTAP-binding motif ( P = 1e-14); middle panel, METTL3-binding motif (P = 1e-13); lower panel, binding motif obtained when only genes found in both WTAP- and METTL3- binding clusters were included (P = 1e-19). Binding motifs were computed by the HOMER program. (D) Venn diagram of the overlapping genes with binding clusters of WTAP and METTL3 in the PAR-CLIP samples. (E) Percentage of WTAP/METTL3 clusters in CDS and UTR regions overlapped with m6A sites. (F) HeLa cells were transfected with siCTRL or siWTAP and Myc-METTL3 for 48 h as indicated. The cell lysates were then subjected to PAR-CLIP using anti-Myc. The pulled down RNA products in the RNA-METTL3 complex were labeled by Biotin and detected by Biotin chemiluminescent nucleic acid kit. (G) Percentage of WTAP- (711 multi-isoform and 41 single-isoform) and METTLE3- (3 155 multi-isoform and 192 single-isoform) binding mRNAs derived from single-isoform or multi-isoform genes and the reference Ensembl genes of human (P = 2.2e- 16, Fisher test). (H) Percentage of constitutively or alternatively spliced exons adjacent to intronic binding clusters of WTAP (left), METTL3 (middle), overlap of WTAP and METTL3 (right). Supportive data were included in Supplementary information, Figures S4 and S5. www.cell-research.com | Cell Research npg WTAP as m6A methyltransferase regulatory subunit genes bound by the two proteins (Figure 3D), 99 genes that the WMM complex might play an inhibitory role in muscle development. Although the proportion of apop- were overlapped (Supplementary information, Figure S5E). Furthermore, assigning genes with METTL3- or totic cells detected by the TUNEL assay was slightly increased in individual morphants, double knockdown of WTAP-binding clusters to either single- or multi-isoform genes revealed that the majority of mRNA species bound WTAP and METTL3 led to a striking increase in apop- tosis (Figure 4C), consistent with observations in human by WTAP or METT3 were derived from multi-isoform genes (Figure 3G; P = 2.2e-16, Fisher test). Refining this cells (Supplementary information, Figure S6D-S6F). The apoptotic cell death observed in WTAP MO-treated observation further, assignment of all intronic clusters to either constitutively or alternatively spliced exons re- zebrafish embryos was rescued by either full-length or an N-terminal region of zebrafish WTAP (Figure 4D). Con- vealed that these clusters are significantly closer to alter - natively spliced exons compared to constitutively spliced sistent with their role in mammalian cells, both METTL3 and WTAP are required for the m6A methyltransferase exons (Figure 3H, P = 2.2e-16, Fisher test), further in- dicating a role of the WMM complex and mRNA m6A activity in zebrafish embryos (Supplementary informa- tion, Figure S6G). These data suggests that the m6A modification in RNA splicing as proposed previously [9]. methyltransferase is required for normal tissue differen- tiation in zebrafish. The WMM complex is required for tissue differentiation in zebrafish The important role of the WMM complex in the regu- Discussion lation of m6A modification, gene expression and mRNA processing in cultured cells prompted us to investigate METTL3 homologs have been found in various spe- its functions at the organismal level using zebrafish em- cies including S. cerevisiae (IME4), Drosophila (Ime4) bryos as the model system. Human and zebrafish WTAP and Arabidopsis (MTA) [23-26, 42]. The two consensus proteins share 81% identity with a high degree of conser- methyltransferase motifs CM I and CM II are present in vation in N-terminal region (Supplementary information, all homologs. Single amino acid substitutions in the puta- Figure S5F). Whole-mount in situ hybridization (WISH) tive catalytic domain of Ime4 lead to loss of m6A modifi- showed that WTAP and METTL3 were maternally ex- cation in mRNA and severe sporulation defects [42, 43]. pressed from the 4-cell stage and ubiquitously expressed In Arabidopsis, MTA is mainly expressed in dividing through early embryogenesis, with enriched expression tissues, particularly in reproductive organs, shoot meri- in the brain region at 36 hpf (hours post fertilization) stems and emerging lateral roots. Inactivation of MTA (Supplementary information, Figure S6A). To determine results in m6A modification defects and developmental whether WTAP and METTL3 are functionally involved failure beyond the globular stage [26]. in early embryonic development, WTAP and METTL3 Despite the identification of a multi-subunit complex were knocked down in zebrafish embryos using antisense possessing m6A methyltransferase activity more than morpholinos (MOs). Embryos injected with WTAP MO 15 years ago, METTL3 has remained the only known displayed multiple developmental defects at 24 hpf in- component of the mammalian complex [22]. There- cluding smaller head and eyes, smaller brain ventricle, fore, unveiling proteins associated with METTL3 in and curved notochord, while embryos injected with the methyltransferase complex is of key importance for METTL3 MO were only modestly affected (Figure 4A). understanding how m6A modifications in RNA might However, simultaneous knockdown of these two genes impact and regulate cellular functions. Previously, At- caused much more severe defects in embryos compared FIP37 and Mum2 were found to associate with plant and to parallel control and embryos injected with individual yeast METTL3 homologs, respectively [26, 28]. Like MOs (Figure 4A). GFP expression of the constructs of MTA, disruption of AtFIP37 also results in developmen- GFP-WTAP and GFP-METTL3 was markedly decreased tal arrest at the globular stage [26, 44]. In the present by co-injection of corresponding MO, verifying efficient study, we have identified WTAP, the human homolog of knockdown (Supplementary information, Figure S6B). AtFIP37/Mum2, and a novel protein METTL14 as two WISH showed that the expression of neuro-ectoderm new components of the human m6A methyltransferase marker goosecoid, and mesoderm marker no tail at the complex. Like METTL3, METTL14 contains a catalytic 50% epiboly stage was not changed, suggesting normal domain and harbors catalytic activity [30]; while our data germ layer formation in the morphants (Supplementary suggest that WTAP likely have very important regulatory information, Figure S6C). However, the expression of so- functions, including tethering the m6A methyltransfer- mite marker myod was increased in WTAP-, METTL3-, ase complex to RNA and facilitating its accumulation and double knockdown embryos (Figure 4B), indicating in nuclear speckles (Figures 2B, 2C and 3F). The fact Cell Research | Vol 24 No 2 | February 2014 npg Xiao-Li Ping et al . Figure 4 The WMM complex plays essential roles during zebrafish embryogenesis. (A) Embryos injected with individual (WTAP-, METTL3-) or combined (METTL3+WTAP) MOs. The double knockdown showed more severe morphological defects, compared to other groups, at 24 hpf. Red arrows mark head and eyes, while blue arrows mark brain ventricle. The curve of the notochord is labeled by the double dashed lines. (B) Expression of somite marker myod in the morphants at 24 hpf. myod expression was increased in WTAP-, METTL3-, or double morphants. (C) Increased apoptosis (TUNEL assay) was observed in embryos injected with individual or combined MOs. (D) Overexpression of full-length or N-terminal but not C-terminal ze- brafish WTAP mRNA prevented apoptosis in zebrafish WTAP-MO embryos. Note that there is auto-fluorescence on the yolk. Anterior to the left and dorsal is up. Scale bar, 250 µm. Supportive data were included in Supplementary information, Figures S5F and S6. that m6A demethylases also localize in nuclear speck- ribonucleotides [2]. Motif analyses of m6A peaks en- les strongly suggests that a dynamic regulation of m6A riched in both human and mouse cells have revealed that levels in mRNA may occur in nuclear speckles [3, 14]. m6A modification occurs in highly conserved regions Furthermore, our studies in zebrafish revealed that MET - with a consensus sequence RRACH (R = G or A; H= A, TL3 mRNA expression level changed during embryonic C or U) [9-13]. The m6A modification occurs in intra - development (also observed in vitro in bovine embryos genic regions particularly in the 3′-end of CDS and the [45]), and that depletion of WTAP and METTL3 com- first quarter of the 3′-UTR [9, 10]. Our PAR-CLIP data promised tissue differentiation (Figure 4 and Supplemen- demonstrate that the conserved WTAP- and METTL3- tary information, Figure S6), strongly suggesting that binding motifs both contain the consensus m6A sequence m6A may play a key role in regulating organismal devel- (Figure 3C), and their binding clusters in CDS and UTR opment. regions significantly overlap with m6A sites (Figure On average, there is 1 m6A modification per 2 000 3E), strongly suggesting a direct connection between the www.cell-research.com | Cell Research npg WTAP as m6A methyltransferase regulatory subunit WMM complex and m6A modification. Interestingly, without normalization of the overall length of the various transcript regions, the majority of WTAP- or METTL3-binding clusters were located in introns (Supplementary information, Figure S4A), which suggests that WMM-dependent m6A modification may be coupled to gene transcription as previously proposed [1-3]. For the relationship of m6A and mRNA splicing, Dominissini and colleagues calculated the average num- ber of m6A peaks in multi-isoform and single-isoform genes, and assigned all m6A peaks to either constitu- Figure 5 A schematic model of the WMM complex function. tively or alternatively spliced exons based on the list of The WMM complex mediates methylation of internal adenosine splicing events of all coding Ensembl genes. Their study residues in eukaryotic mRNA, forming N6-methyladenosine. revealed that m6A modification is significantly over-rep - WTAP binds to the m6A consensus RRACH motif of mRNA and resented in multi-isoform genes and alternatively spliced recruits catalytic subunits METTL3 and METTL14. Then the exons, proposing that m6A may affect RNA splicing [9]. METTL3-METTL14 complex carries out m6A methyltransferase activity in the m6A motif. Consistent with this observation, we found an enrichment of WTAP/METTL3-binding clusters in genes with multi- isoforms and alternatively spliced exon junctions based on our PAR-CLIP+RNA-seq dataset (Figure 3G and 3H). Materials and Methods Taken together, these results suggest that WTAP/MET- TL3 and thereby the m6A methyltransferase may influ - Cell culture, plasmids and antibodies ence RNA splicing. However, a thorough understanding Human cells were cultured in standard cell culture Dulbecco’s of the relationship between m6A and transcription and/or modified Eagle’s medium at 37 °C in a humidified incubator with splicing awaits future investigations. 5% CO (v/v).The human METTL3 gene was cloned into pCMV- Myc (Invitrogen), S-protein/FLAG/SBP (streptavidin-binding Drosophilia female-lethal (2)D (FL (2)D, WTAP protein) triple-tagged destination vector [48] or pGEX-5×-2 (GE homolog) also interacts with the female-specific RNA- healthcare). The human WTAP gene was cloned into p3XFLAG- binding protein sex-lethal (SXL) [46, 47], VIR [46], Snf, CMV-14 (Sigma) or pProEX-HTb (Invitrogen). The human MET- U2AF, U2A50 and U2AF38 [47]. This together with the TL14 gene was cloned into pcDNA3-HA (Invitrogen). The follow- results showing that there is a difference in the numbers ing antibodies were used: rabbit-anti-METTL3 (Abcam), mouse- of genes containing METTL3- or WTAP-binding clus- anti-METTL3 (Abnova), mouse-anti-WTAP (Santa Cruz), rabbit- ters might suggest that METTL3 is the core component anti-METTL14 (Atlas), rabbit-anti-m6A (Synaptic Systems) and other antibodies included in Supplementary information, Data S1. of the m6A methyltransferase, whereas WTAP serves as its regulatory subunit. Based on the findings that WTAP Tandem affinity purification and mass spectrometry interacts directly with METTL3 (Figure 1) and medi- 293T cells transiently transfected with triply-tagged METTTL3 ates nuclear speckle localization of both METTL3 and plasmid (SFB-METTL3) were used for tandem affinity purifica - METTL14, we propose a model, in which METTL3 and tion. Cell lysates were incubated with streptavidin-Sepharose METTL14 are anchored to mRNA by WTAP, allow- beads (Amersham Biosciences), and eluted with 1 mg/ml bio- ing subsequent methylation of target adenosine residues tin (Sigma). The eluates were further incubated with S-protein- agarose (Novagen). The proteins bound to S-protein-agarose beads (Figure 5). were subjected to SDS-PAGE and visualized by coomassie blue In summary, our findings identify WTAP as a regula - staining. The identities of eluted proteins were revealed by mass tory subunit required for formation of a functional m6A spectrometry analysis. The Q Exactive mass spectrometry data methyltransferase complex including METTL3 and (Thermo Fisher Scientific) were searched against SwissProt hu- METTL14, which play an important role in the regula- man database using 15 ppm peptide mass tolerance and 20mmu tion of gene expression and alternative splicing. Thus, fragment mass tolerance. the discovery and characterization of the RNA m6A methyltransferase complex, together with the recent ad- Immunoprecipitation 293T cells transfected with the indicated siRNAs and/or DNA vances in the understanding of the demethylases FTO constructs were lysed in buffer (100 mM NaCl, 20 mM Tris- and ALKBH5, is an important step towards a more thor- HCl (pH 7.4), 0.5% NP-40, 1 mM PMSF, 1 mM Na VO 1 mM 3 4, ough understanding of the biological significance of the β-glycerophosphate, 1 mM NaF and 1× Cocktail), and subjected epitranscriptomic marker m6A. to immunoprecipitation (IP) followed by immunoblotting with the indicated antibodies. Cell Research | Vol 24 No 2 | February 2014 npg Xiao-Li Ping et al . The following antibodies were used in the study: mouse-anti- to the size of Myc-METTL3 or Flag-WTAP. Purified RNAs from Flag (Sigma), rabbit-anti-Myc (Abcam), rabbit-anti-HA(Clontech), RNA-Protein complex were subjected to the small RNA library rabbit-anti-WTAP (Atlas), Anti-Rabbit IgG-HRP (Dakocytoma- construction and then sequenced with HiSeq 2000 system (Illumina tion), Anti-Mouse IgG-HRP (Dakocytomation). Inc.). Bowtie 0.12.7 software [52] was applied to map sequencing reads against human (hg19) genome with up to two mismatches allowed. To multi-mapped reads, mapped locations were only GST pull-down assay The human METTL3 gene was subcloned into pGEX-5X-2 reported for those with the minimum number of observed mis- expression plasmid with GST-tag and the human WTAP gene matches. PARalyzer software [36] was used to define METTL3- was subcloned into pProEX-HTb expression plasmid with His and WTAP-binding sites. Sequence motifs enriched in clusters tag. Recombinant GST-METTL3 and His-WTAP proteins were were identified by HOMER, a suite of tools for motif discovery induced to express in E. coli strain BL21(DE3) and purified by and next-generation sequencing analysis [38]. FPLC using Bio-Scale Mini Profinity GST cartridge (Biorad) and Total RNA was extracted from 293T cells transfected with Bio-Scale Mini Profinity IMAC cartridge (Biorad) as described siRNA using RNAzol (Molecular Research Center, Inc., USA). in the commercial instruction manual. The quality of proteins was cDNA library was constructed using TruSeq RNA Sample Prep tested by western blot and coomassie brilliant blue staining. His- Kit and then sequenced with HiSeq 2000 system (Illumina Inc.). WTAP proteins were incubated with glutathione sepharose (GE RNA-seq reads were aligned against the Ensembl genome (hg19) Healthcare) to be pre-cleared in NETN buffer (100 mM NaCl, 1 using TopHat2 [40, 53]. The number of reads mapped to each of mM EDTA, 20 mM Tris-HCl pH 7.4, 0.5% NP-40). Then pre- the Ensembl genes (version 72) was counted using the HTSeq cleared His-WTAP was mixed with GST or GST-METTL3 protein python package [54]. DEGs were determined using the R-package and incubated overnight at 4 °C with equal amount of glutathione DEGseq with the method MARS (MA-plot-based method with sepharose beads. After washing the beads five times with NETN random sampling model), P value cutoff = 0.001 [55]. GO analy- buffer, proteins bound to the beads were analyzed by 8% SDS- ses of DEGs and PAR-CLIP data were performed using DAVID PAGE followed by immunoblotting with HRP-conjugated anti-His [56, 57]. Enrichment map of DEGs was constructed by Cytoscape and anti-GST antibodies (Abcam). 2.8.3 software [57]. The FPKM (Fragments Per Kilobase of exon per Million mapped reads) value for each transcript was calculated by using Cufflinks version 2.0.0 and the differentially expressed Immunofluorescence staining HeLa cells grown on coverslips were transfected with siRNA transcripts were identified by Cuffdiff program in the Cufflinks (50 nM) or DNA constructs as indicated for about 4 h, and 24 h software package [58]. Scripture, a method for transcriptome re- later fixed with 4% paraformaldehyde on ice followed by permea - construction that relies solely on RNA-Seq reads and an assembled bilization with 0.1% Triton X-100 and 0.05% NP-40 on ice. After genome to build the transcriptome [59], was applied to calculate pre-blocking with 5% non-fat dried milk in TBST, coverslips were the isoform number for each gene. Variation in the isoform number first incubated with primary antibody for 1 h and then with fluo- between METTL3/WTAP-depleted and control samples indicated rescent dye-conjugated secondary antibody for 0.5 h and mounted an alternative splicing event. with DAPI-containing mounting medium (Vector Laboratories, Furthermore, a Bowtie library of non-redundant set of se- Burlingame, CA, USA) and visualized under immunofluorescence quences consisting of all possible junctions between the exons of confocal microscope Leica TCS SP5 (Leica). The method used each Refseq gene of human was generated and all the RNA-seq for immunofluorescence staining following RNase A treatment reads were aligned against this library with Bowtie 0.12.7 software was adapted from Mayer et al. [49] and Sytnikova et al. [50]. In [52]. To the exon-exon junctions (EEJs) with read mapping, ac- brief, cells grown on coverslips were permeabilized with 0.05% cording to their continuous or discrete exon number, they can be Triton X-100 in 20 mM Tris-HCl (pH 7.4), 5 mM MgCl , 0.5 mM divided into constitutively or alternatively spliced exons. All the EDTA, and 25% glycerol; washed twice with PBS; and treated WTAP/METTL3 clusters were assigned to these exons. with RNase A (1 mg/ml) (diluted in PBS) in 37 ºC for 7 min. After washing, cells were fixed with cold methanol (15 min) and stained MO-mediated gene knockdown in zebrafish embryos with the indicated antibodies following the blocking procedure. The antisense MO oligonucleotides targeting individual mem- The immunofluorescence images were digitally recorded by using bers of the WMM complex were synthesized (Gene Tools, LLC.). confocal microscope. The following antibodies were used in im- The MOs (2.5-8 ng) were injected into embryos from one-cell munofluorescence staining: mouse-anti-Flag (Sigma), rabbit-anti- to four-cell stage. Zebrafish WTAP and METTL3 were amplified Myc (Abcam), rabbit-anti-HA (Clontech), mouse-anti-METTL3 from zebrafish cDNA library by PCR and sequentially subcloned (Abnova), rabbit-anti-METTL14 (Atlas), mouse-anti-WTAP (Santa into the pGEM-T vector and pEGFP-N1 (Clontech) to generate Cruz), mouse-anti-SC35 (Sigma), anti-mouse IgG-Cy3 (Sigma), pWTAP-GFP or pMETTL3-GFP constructs for checking MO ef- anti-rabbit IgG-Cy3 (Sigma), anti-mouse IgG-FITC (Sigma), anti- ficiency rabbit IgG-FITC (Sigma). Statistical analysis PAR-CLIP and RNA-Seq Two-tailed t-tests in grouped analyses of Prism5 software 293T cells expressing Myc-METTL3 or Flag-WTAP were cul- (GraphPad Software Inc., USA) were applied. P-values < 0.05 tured in medium supplemented with 4-SU (Sigma), irradiated with were considered significant. 365 nm UV light to induce crosslinking as described previously [51]. Immunoprecipitated protein-RNA complexes were separated Accession numbers by SDS-PAGE and then transferred to PVDF membrane; RNA- Mass spectrometry data have been uploaded to Peptide Atlas protein complexes were cut out from the membrane corresponding under accession number PASS00296. PAR-CLIP and RNA-Seq www.cell-research.com | Cell Research npg WTAP as m6A methyltransferase regulatory subunit data have been uploaded to GEO database and can be accessed via adenosine sites of HeLa cell messenger ribonucleic acid. Bio- accession number GSE50583. chemistry 1977; 16:1672-1676. 14 Jia G, Fu Y, Zhao X, et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Acknowledgments Chem Biol 2011; 7:885-887. 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Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase

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Springer Journals
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Copyright © 2014 by The Author(s)
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Life Sciences; Life Sciences, general; Cell Biology
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10.1038/cr.2014.3
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Abstract

Cell Research (2014) 24:177-189. npg © 2014 IBCB, SIBS, CAS All rights reserved 1001-0602/14 www.nature.com/cr ORIGINAL ARTICLE Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase 1, 8, * 1, * 2, * 1, 8, * 1 1 1 Xiao-Li Ping , Bao-Fa Sun , Lu Wang , Wen Xiao , Xin Yang , Wen-Jia Wang , Samir Adhikari , 1 1 1 1 1 1 1 3 4 Yue Shi , Ying Lv , Yu-Sheng Chen , Xu Zhao , Ang Li , Ying Yang , Ujwal Dahal , Xiao-Min Lou , Xi Liu , 5 6 6 6 1 4 Jun Huang , Wei-Ping Yuan , Xiao-Fan Zhu , Tao Cheng , Yong-Liang Zhao , Xinquan Wang , 1, 7 2 1, 8 Jannie M Rendtlew Danielsen , Feng Liu , Yun-Gui Yang Center For Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China; State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Chinese Academy of Sciences Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China; Center for Structural Biology, Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China; Life Sciences Institute, Zhejiang University, Zhejiang 310058, China; State Key Laboratory of Experimental Hematology, Institute of Hematology, Tian- jin 300041, China; The Novo Nordisk Foundation Center for Protein Research, Ubiquitin Signalling Group, Faculty of Health Sciences, Copenhagen, Denmark; University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms’ tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic ac- tivity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-en- hanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransfer - ase complex and play a critical role in epitranscriptomic regulation of RNA metabolism. Keywords: WTAP; m6A methyltransferase; METTL3; METTL14; mRNA Cell Research (2014) 24:177-189. doi:10.1038/cr.2014.3; published online 10 January 2014 Adomet) serving as the methyl donor [1-3]. The N6- Introduction methyladenosine (m6A) modification is one of the most Nitrogen or oxygen atoms in RNA are ubiqui - frequent methylations in eukaryotic mRNA, accounting tously methylated with S-adenosylmethionine (SAM, for over 80% of all RNA base methylations and, it has been observed in various species [4-8]. Recently, two independent studies combining m6A immunoprecipita- *These four authors contributed equally to this work. tion with high-throughput analysis revealed that the m6A a b Correspondence: Yun-Gui Yang , Feng Liu modification tends to mainly occur in intragenic regions. E-mail: [email protected] The frequency is particularly high in the 3′-end of cod- E-mail: [email protected] ing sequence (CDS) and the first quarter of the 3′-UTR, Received 13 October 2013; revised 3 December 2013; accepted 4 Decem- ber 2013; published online 10 January 2014 with an average of 1 m6A per 2 000 ribonucleotides. The npg WTAP as m6A methyltransferase regulatory subunit m6A modification occurs in highly conserved regions 28]. METTL14 protein shares 43% identity (Supple- with the consensus sequence, RRACH (R = G or A; H = mentary information, Figure S1B) with METTL3, and A, C or U) [9-13]. During brain development a dynamic a previous phylogenetic analysis has suggested that the change in m6A levels on RNA has been observed [10], two proteins are homologs [23]. Furthermore, METTL14 suggesting the existence of RNA m6A demethylase(s). contains similar motifs essential for catalytic activity as Indeed, two AlkB dioxygenase family proteins, fat mass METTL3 (Supplementary information, Figure S1B). The and obesity associated gene (FTO) and ALKBH5, were interactions among METTL3, WTAP and METT14 were recently demonstrated to catalyze the removal of the confirmed by co-immunoprecipitation (Figure 1A, 1E m6A mark both in vitro and in vivo [14, 15]. These two and Supplementary information, Figure S2A and S2B). enzymes function in various biological processes such The specificity of the interaction between METTL3 and as development, metabolism, and fertility. They regulate WTAP was verified by examination of the endogenous the expression levels of thousands of genes, suggesting a interaction between these two proteins after depletion pivotal epitranscriptomic function of m6A modification of WTAP by small interfering RNAs (siRNA) (Figure in regulating RNA metabolism [8, 16-19]. 1B). GST pull-down using purified recombinant GST- In contrast to the discovery of RNA m6A demethyl- METTL3 and 6×His-WTAP proteins demonstrated a di- ases, the nature of the methyltransferase complex respon- rect interaction between METTL3 and WTAP (Figure 1C sible for catalyzing m6A formation in RNA still remains and Supplementary information, Figure S1C). By using largely unknown. The mammalian m6A methyltransfer- WTAP constructs with either the C- (WTAP-N, 1-200 ase complex is generally perceived to contain at least two aa) or N-terminus (WTAP-C, 201-396 aa) truncated, we key subcomplexes designated MT-A (200 kD) and MT-B found that the interaction between WTAP and METTL3 (800 kD) [20, 21]. So far only a 70-kD protein, methyl- or METTL14 depends on the N-terminus of WTAP (Fig- transferase like 3 (METTL3) has been identified as one ure 1D and 1F). It has been reported that RNA is not component of the 200-kD MT-A complex. METTL3 ho- an essential component of the m6A methyltransferase mologs have been identified in S. cerevisiae, Drosophila complex [20, 29]. Consistently, we observed that nei- and Arabidopsis [22-26]. Although mRNA methylation ther RNase A treatment nor inhibition of methylation by occurs primarily within the RRACH consensus sequence cycleoleucine affected the interaction between WTAP in mammals, only a portion of the RRACH sites contain and METTL3 (Supplementary information, Figure S2C- the m6A modification [9, 10, 27]. Mechanistic under- S2E). Based on these findings, we conclude that WTAP standing of the selectivity, regulation and function of the and METTL14 are bona fide components of the m6A m6A modification depends on a complete characteriza- methyltransferase complex, and that both RNA and the tion of the methyltransferase complex. m6A modification are dispensable for the interaction In this study, we identify for the first time, Wilms’ between WTAP and METTL3. In the remaining of this tumor 1 (WT1)-associating protein (WTAP) and meth- paper, we will refer to this complex as the WMM (WTAP, yltransferase like 14 (METTL14) as components of the METTL3 and METTL14) complex. mammalian m6A methyltransferase complex. In particu- lar, WTAP appears to serve as a regulatory subunit essen- WTAP is required for m6A methyltransferase activity in tial for m6A methyltransferase activity. vivo WTAP does not harbor any obvious catalytic domains, and in contrast to METTL3 and METTL14 that were Results recently demonstrated to form an active complex capable Identification of WTAP and METTL14 as METTL3-asso - of catalyzing m6A formation, WTAP showed no catalytic activity itself or effect on the activity of METTL3-MET- ciating proteins TL14 complex in vitro [30]. Given the direct interaction To identify mammalian METTL3-associating pro- between WTAP and METTL3, we speculated that WTAP teins, we utilized tandem affinity purification to pull may serve to regulate m6A methyltransferase activity down stably expressed SFB-tagged METTL3 from 293T in vivo. To test this hypothesis, we examined the m6A cells. Subsequent mass spectrometry analysis of affinity- levels in mRNA extracted from WTAP- or METTL3- purified SFB-METTL3 protein complexes identified knockdown cells. Dot-blot assays showed that depletion two potential METTL3-associating proteins, WTAP and of either of these proteins resulted in significantly lower METTL14 (Supplementary information, Figure S1A). m6A levels in mRNA (Figure 2A and Supplementary in- Consistently, AtFIP37 and Mum2, the WTAP homologs formation, Figure S3A). RT-PCR and western blot analy- in plant and yeast, respectively, have previously been ses verified the knockdown efficiency and specificity, shown to associate with METTL3 in these species [26, Cell Research | Vol 24 No 2 | February 2014 npg Xiao-Li Ping et al . Figure 1 WTAP interacts with METTL3 and METTL14. (A) 293T cells were transfected with Flag-WTAP and Myc-METTL3 constructs as indicated. Forty-eight hours later, cells were lysed and the lysates were subjected to immunoprecipitation using anti-Myc (Myc-IP) followed by immunoblotting with the anti-Flag antibodies. (B) 293T cells were treated with control siRNA (siCTRL) or siRNA targeting WTAP (siWTAP) for 48 h. Then cells were lysed and the lysates were subjected to IP using anti- WTAP. The immunoprecipitated samples were analyzed by immunoblotting with the anti-METTL3 antibodies. (C) Purified recombinant His-WTAP proteins were mixed with either GST or GST-METTL3 proteins as indicated, pulled down with GST beads, and subjected to immunoblotting with the indicated antibodies. (D) 293T cells were co-transfected with Myc-METTL3 and Flag-WTAP full-length (-FL), N-terminal (-N) or C-terminal (-C) constructs as indicated. Forty-eight hours later, cells were lysed and the lysates were subjected to Myc-IP followed by immunoblotting with the anti-Flag antibodies. (E) 293T cells were transfected with Flag-WTAP and HA-METTL14 constructs as indicated. Forty-eight hours later cells were lysed and the ly- sates were subjected to HA-IP followed by immunoblotting with the anti-Flag antibodies. (F) 293T cells were co-transfected with HA-METTL14 and Flag-WTAP full-length (-FL), N-terminal (-N) or C-terminal (-C) constructs as indicated. Forty-eight hours later, cells were lysed and the lysates were subjected to HA-IP followed by immunoblotting with the anti-Flag antibod- ies. Supportive data were included in Supplementary information, Figures S1 and S2. www.cell-research.com | Cell Research npg WTAP as m6A methyltransferase regulatory subunit respectively (Supplementary information, Figure S3B role in regulating m6A modification is independent of and S3C). Moreover, depletion of WTAP or METTL3 its previously described binding partner WT1 (Figure did not affect the expression of the other complex com- 2A and Supplementary information, Figure S3A). Taken ponents (Supplementary information, Figure S3B and together, these findings suggest that WTAP affects m6A S3C). WTAP was originally identified as a protein asso - methyltransferase activity in vivo. ciated with WT1 [31]. We examined whether WT1 might also be required for m6A methyltransferase activity in WTAP r egulates accumulation of METTL3 and vivo. Interestingly, we found that WT1 depletion had no METTL14 in nuclear speckles influence on m6A abundance, suggesting that WTAP’s Previous studies have indicated that m6A modification Figure 2 WTAP regulates the nuclear speckle localization of METTL3 and METTL14. (A) The graph represents the quantifi - cation of three independent dot-blot experiments (raw data were included in Supplementary information, Figure S3A). The y- axis represents the relative intensity of dots relative to that of the control group. P values were calculated using a two-tailed t- test. Error bars represent SD. (B-D) After transfection (48 h) with the indicated fluorescence FAM-labeled siRNA, HeLa cells were fixed and immunostained with the indicated antibodies. DNA was visualized by DAPI. Scale bar, 10 μm. Supportive data were included in Supplementary information, Figure S3. Cell Research | Vol 24 No 2 | February 2014 npg Xiao-Li Ping et al . occurs at the pre-mRNA stage within the nucleus and it and fixation procedures. A similar staining pattern in nuclear speckles has also previously been reported [15]. thereby may affect splicing and mRNA export [2, 32- 35]. Nuclear speckles are enriched in proteins involved mRNA is the major RNA species bound by WTAP and in RNA processing and alternative splicing. Interest- ingly, METTL3 and WTAP as well as the m6A demeth- METTL3 We next performed Photoactivatable-Ribonucleoside- ylases FTO and ALKBH5 have previously been shown to colocalize with nuclear speckle markers [14, 15, 31]. Enhanced Crosslinking and Immunoprecipitation (PAR- CLIP) [51] to identify the targets of WTAP and METTL3 The physical interaction between WTAP and METTL3 and METTL14 prompted us to examine the cellular proteins. In brief, 4-thiouridine (4-SU), a photoreactive ribonucleoside analog, was incorportated into RNA tran- distribution of METLL14. Using immunofluorescence microscopy, we found that endogenous METTL14, like scripts, which leads to the thymidine-to-cytidine muta- tion in crosslinked sequences. Therefore, one unique METTL3 and WTAP, exhibits a diffused nucleoplasmic staining pattern with intense granule-like foci, and that feature of cDNA libraries prepared by PAR-CLIP is that the precise position of crosslinking can be identified by it colocalizes well with the pre-mRNA splicing factor SC35 in nuclear speckles (Figure 2C and Supplementary mutations residing in the sequenced cDNA. PARalyzer [36, 37] software was used to identify the preferred re- information, Figure S3F). Interestingly, WTAP deple- tion decreased the accumulation of both METTL3 and gions bound by WTAP and METTL3. 4 986 METTL3- and 922 WTAP-binding clusters were enriched and could METTL14 in nuclear speckles (Figure 2B, 2C and Sup- plementary information, Figure S3G), whereas knock- be assigned into four RNA categories: mRNA, miRNA, large intergenic non-coding RNA (lincRNA) and pseu- down of either METTL3 or METTL14 had no effect on the localization of WTAP (Figure 2D and Supplementary dogene (Figure 3A). About 95% and 92% of the RNA bound by METTL3 and WTAP, respectively, represented information, Figure S3G). Furthermore, METTL3 deple- tion affected the nuclear speckle localization of MET- mRNA, demonstrating that mRNA is the major substrate of the WMM complex. We further mapped the mRNA TL14 and vice versa (Figure 2B and 2C). Consistent with our observations on m6A methyltransferase activity, clusters into four non-overlapping transcript regions, 5′- UTR, CDS, 3′-UTR and intron, according to the human WT1 depletion did not affect METTL3 or WTAP local- ization (Figure 2B and 2D). Based on these observations, reference transcript annotation. After normalization of the number of clusters to the overall length of the dif- we conclude that WTAP is required for accumulation of METTL3 and METTL14 in nuclear speckles. ferent transcript regions, the majority of binding sites of WTAP and METTL3 were identified in CDS and UTR Nuclear speckle-associating factors can be classified as RNase-sensitive or -insensitive factors based on the regions (Figure 3B). To define the in vivo RNA recognition elements for nature of the association with the nuclear speckles. To characterize the nature of the association of the WMM WTAP and METTL3, the binding cluster data were ana- lyzed by HOMER, a suite of tools for motif discovery complex with nuclear speckles, we treated cells with RNase A. Similar to the mRNA processing factor SM and next-generation sequencing analysis [38]. In this analysis procedure, the WTAP and METTL3 clusters (smith antigen), but unlike the splicing factor SC35, RNase A treatment resulted in a dramatic decrease of were set as the target sequences, and a set of background clusters was generated with the BEDTools’ shuffleBed WMM complex accumulation in nuclear speckles, dem- onstrating that the WMM complex associates with RNA, program [39] to randomly shuffle regions of the same size as the clusters throughout the gene regions, with which is essential for the recruitment and/or retention of the WMM complex in nuclear speckles (Supplementary the parameter for HOMER motif length from 5 to 8. The motifs AGGACU (P = 1e-14) and UGUGGACU information, Figure S3D-S3F). It is worth noting that METTL3 and METTL14 staining almost disappeared (P = 1e-13) were enriched in WTAP- and METTL3- binding clusters, respectively (Figure 3C). When we only upon WTAP knockdown, but the overall protein levels of METTL3 and METTL14 did not change following included genes found in both WTAP- and METTL3- binding clusters, the highest scoring motif was UUAG- WTAP depletion (Supplementary information, Figure S3B and S3C). It is likely that when WTAP is depleted, GACU (P = 1e-19) (Figure 3C). In addition, AGGAC METTL3 and METTL14 no longer bind to RNA in the (P = 1e-12), UGGAC (P = 1e-12), and AGACUAA (P nuclear speckles, and become diffused within the entire = 1e-10) were also highly enriched in WTAP, METTL3 cell, which reduces the staining density to levels below and WTAP/METTL3 overlay clusters, respectively (Fig- detection. In addition, unbound METTL3 and METTL14 ure 3D and Supplementary information, Figure S4B). might easily get washed out during the permabilization This is in accordance with the reported consensus m6A www.cell-research.com | Cell Research npg WTAP as m6A methyltransferase regulatory subunit Cell Research | Vol 24 No 2 | February 2014 npg Xiao-Li Ping et al . motif RRACH (R = G or A; H = A, C or U) [9, 10]. The Taken together, these findings support the existence of high degree of similarity of the mRNA binding motifs a WTAP-METTL3-containing complex that methylates between WTAP and METTL3 are consistent with the re- RNA at specific sites in mammals. sults that WTAP and METTL3 form a complex. We next calculated the distance between WTAP/ The WMM complex regulates transcription and METTL3 clusters and the m6A sites. To obtain the best alternative splicing coverage of m6A sites, we downloaded human m6A data Gene Ontology (GO) analyses of mRNA captured by released by Dominissini et al. [9] and Meyer et al. [10] METTL3 and WTAP using PAR-CLIP revealed that a and aligned the data to the hg19 genome by Tophat2 [40]. large proportion of mRNA species associated with both We then used MACS [41] and Fisher test [9, 10] meth- proteins were derived from genes involved in transcrip- ods to determine m6A peaks. The m6A peaks identified tion and RNA metabolism/splicing (Supplementary in this analysis were combined with the previously pub- information, Figure S5A). To further investigate the po- lished m6A sites, and the duplicates were removed. Then tential roles of the WMM complex in RNA metabolism, we used the BEDTools’ closestBed [39] to calculate the transcriptome analyses were performed to compare the distance between WTAP/METTL3 clusters and m6A genome-wide gene expression and alternative splicing sites. We generated control clusters with BEDTools’ events between wild-type cells and WTAP- or METTL3- shuffleBed [39] to randomly shuffle regions of the same deficient cells. The transcriptome analyses revealed a sig- nificant overlap in differentially expressed genes (DEGs) size as the clusters. The result demonstrated a significant spatial correlation (P = 2.2e-16) with 74% WTAP- and between WTAP- and METTL3-deficient cells (Supple- mentary information, Figure S5C). Consistent with the 69% METTL3-binding clusters in CDS and UTR regions overlapping with m6A sites (Figure 3E and Supplemen- GO analysis of the PAR-CLIP data, GO analysis of the overlapping DEGs from WTAP- and METTL3-deficient tary information, Figure S4C and S4D). These results strongly suggest a significant role of both WTAP and cells demonstrated that most of these genes were related to transcription and RNA processing (Supplementary METTL3 in m6A modification. The results that WTAP is required for nuclear speckle information, Figure S5D), further emphasizing the poten- tial significance of WTAP and METTL3 in RNA metabo - localization of METTL3 and METTL14 and for m6A modification prompted us to test whether the absence of lism. Interestingly, both METTL3 and WTAP knockdowns WTAP might influence the binding of METTL3 to RNA. Interestingly, we found a significantly reduced amount resulted in a pronounced variation in isoform numbers with about half of the variations (2 296) present in both of RNA associated with METTL3 upon WTAP depletion (Figure 3F and Supplementary information, Figure S5B), WTAP- and METTL3-deficient cells (Supplementary information, Figure S5E). When comparing the 2 296 suggesting that WTAP is likely responsible for recruit- ing the m6A methyltransferase complex to target RNAs. genes with changes in isoform numbers with the 410 Figure 3 METTL3 and WTAP bind m6A consensus sequence in mRNA and affect gene expression and alternative splicing. (A) Percentage of various RNA species bound by WTAP and METTL3 based on PAR-CLIP analyses. The WTAP- and METTL3- binding clusters were identified by PARalyzer algorithm. Annotation of clusters was based on the human Ensembl gtf file (ver- sion 72, hg19). The majority of binding sites of WTAP and METTL3 were located in mRNA. (B) Pie chart depicting the distri- bution of binding clusters in mRNA based on PAR-CLIP sequence clusters for WTAP and METTL3 after normalization of the overall length of the different transcript regions. Length of these different transcript regions was extracted from Ensembl an- notations and the distribution percentage of clusters in these regions were normalized by their length. (C) Enriched sequence motif analysis of binding clusters indentified by PAR-CLIP. Upper panel, WTAP-binding motif ( P = 1e-14); middle panel, METTL3-binding motif (P = 1e-13); lower panel, binding motif obtained when only genes found in both WTAP- and METTL3- binding clusters were included (P = 1e-19). Binding motifs were computed by the HOMER program. (D) Venn diagram of the overlapping genes with binding clusters of WTAP and METTL3 in the PAR-CLIP samples. (E) Percentage of WTAP/METTL3 clusters in CDS and UTR regions overlapped with m6A sites. (F) HeLa cells were transfected with siCTRL or siWTAP and Myc-METTL3 for 48 h as indicated. The cell lysates were then subjected to PAR-CLIP using anti-Myc. The pulled down RNA products in the RNA-METTL3 complex were labeled by Biotin and detected by Biotin chemiluminescent nucleic acid kit. (G) Percentage of WTAP- (711 multi-isoform and 41 single-isoform) and METTLE3- (3 155 multi-isoform and 192 single-isoform) binding mRNAs derived from single-isoform or multi-isoform genes and the reference Ensembl genes of human (P = 2.2e- 16, Fisher test). (H) Percentage of constitutively or alternatively spliced exons adjacent to intronic binding clusters of WTAP (left), METTL3 (middle), overlap of WTAP and METTL3 (right). Supportive data were included in Supplementary information, Figures S4 and S5. www.cell-research.com | Cell Research npg WTAP as m6A methyltransferase regulatory subunit genes bound by the two proteins (Figure 3D), 99 genes that the WMM complex might play an inhibitory role in muscle development. Although the proportion of apop- were overlapped (Supplementary information, Figure S5E). Furthermore, assigning genes with METTL3- or totic cells detected by the TUNEL assay was slightly increased in individual morphants, double knockdown of WTAP-binding clusters to either single- or multi-isoform genes revealed that the majority of mRNA species bound WTAP and METTL3 led to a striking increase in apop- tosis (Figure 4C), consistent with observations in human by WTAP or METT3 were derived from multi-isoform genes (Figure 3G; P = 2.2e-16, Fisher test). Refining this cells (Supplementary information, Figure S6D-S6F). The apoptotic cell death observed in WTAP MO-treated observation further, assignment of all intronic clusters to either constitutively or alternatively spliced exons re- zebrafish embryos was rescued by either full-length or an N-terminal region of zebrafish WTAP (Figure 4D). Con- vealed that these clusters are significantly closer to alter - natively spliced exons compared to constitutively spliced sistent with their role in mammalian cells, both METTL3 and WTAP are required for the m6A methyltransferase exons (Figure 3H, P = 2.2e-16, Fisher test), further in- dicating a role of the WMM complex and mRNA m6A activity in zebrafish embryos (Supplementary informa- tion, Figure S6G). These data suggests that the m6A modification in RNA splicing as proposed previously [9]. methyltransferase is required for normal tissue differen- tiation in zebrafish. The WMM complex is required for tissue differentiation in zebrafish The important role of the WMM complex in the regu- Discussion lation of m6A modification, gene expression and mRNA processing in cultured cells prompted us to investigate METTL3 homologs have been found in various spe- its functions at the organismal level using zebrafish em- cies including S. cerevisiae (IME4), Drosophila (Ime4) bryos as the model system. Human and zebrafish WTAP and Arabidopsis (MTA) [23-26, 42]. The two consensus proteins share 81% identity with a high degree of conser- methyltransferase motifs CM I and CM II are present in vation in N-terminal region (Supplementary information, all homologs. Single amino acid substitutions in the puta- Figure S5F). Whole-mount in situ hybridization (WISH) tive catalytic domain of Ime4 lead to loss of m6A modifi- showed that WTAP and METTL3 were maternally ex- cation in mRNA and severe sporulation defects [42, 43]. pressed from the 4-cell stage and ubiquitously expressed In Arabidopsis, MTA is mainly expressed in dividing through early embryogenesis, with enriched expression tissues, particularly in reproductive organs, shoot meri- in the brain region at 36 hpf (hours post fertilization) stems and emerging lateral roots. Inactivation of MTA (Supplementary information, Figure S6A). To determine results in m6A modification defects and developmental whether WTAP and METTL3 are functionally involved failure beyond the globular stage [26]. in early embryonic development, WTAP and METTL3 Despite the identification of a multi-subunit complex were knocked down in zebrafish embryos using antisense possessing m6A methyltransferase activity more than morpholinos (MOs). Embryos injected with WTAP MO 15 years ago, METTL3 has remained the only known displayed multiple developmental defects at 24 hpf in- component of the mammalian complex [22]. There- cluding smaller head and eyes, smaller brain ventricle, fore, unveiling proteins associated with METTL3 in and curved notochord, while embryos injected with the methyltransferase complex is of key importance for METTL3 MO were only modestly affected (Figure 4A). understanding how m6A modifications in RNA might However, simultaneous knockdown of these two genes impact and regulate cellular functions. Previously, At- caused much more severe defects in embryos compared FIP37 and Mum2 were found to associate with plant and to parallel control and embryos injected with individual yeast METTL3 homologs, respectively [26, 28]. Like MOs (Figure 4A). GFP expression of the constructs of MTA, disruption of AtFIP37 also results in developmen- GFP-WTAP and GFP-METTL3 was markedly decreased tal arrest at the globular stage [26, 44]. In the present by co-injection of corresponding MO, verifying efficient study, we have identified WTAP, the human homolog of knockdown (Supplementary information, Figure S6B). AtFIP37/Mum2, and a novel protein METTL14 as two WISH showed that the expression of neuro-ectoderm new components of the human m6A methyltransferase marker goosecoid, and mesoderm marker no tail at the complex. Like METTL3, METTL14 contains a catalytic 50% epiboly stage was not changed, suggesting normal domain and harbors catalytic activity [30]; while our data germ layer formation in the morphants (Supplementary suggest that WTAP likely have very important regulatory information, Figure S6C). However, the expression of so- functions, including tethering the m6A methyltransfer- mite marker myod was increased in WTAP-, METTL3-, ase complex to RNA and facilitating its accumulation and double knockdown embryos (Figure 4B), indicating in nuclear speckles (Figures 2B, 2C and 3F). The fact Cell Research | Vol 24 No 2 | February 2014 npg Xiao-Li Ping et al . Figure 4 The WMM complex plays essential roles during zebrafish embryogenesis. (A) Embryos injected with individual (WTAP-, METTL3-) or combined (METTL3+WTAP) MOs. The double knockdown showed more severe morphological defects, compared to other groups, at 24 hpf. Red arrows mark head and eyes, while blue arrows mark brain ventricle. The curve of the notochord is labeled by the double dashed lines. (B) Expression of somite marker myod in the morphants at 24 hpf. myod expression was increased in WTAP-, METTL3-, or double morphants. (C) Increased apoptosis (TUNEL assay) was observed in embryos injected with individual or combined MOs. (D) Overexpression of full-length or N-terminal but not C-terminal ze- brafish WTAP mRNA prevented apoptosis in zebrafish WTAP-MO embryos. Note that there is auto-fluorescence on the yolk. Anterior to the left and dorsal is up. Scale bar, 250 µm. Supportive data were included in Supplementary information, Figures S5F and S6. that m6A demethylases also localize in nuclear speck- ribonucleotides [2]. Motif analyses of m6A peaks en- les strongly suggests that a dynamic regulation of m6A riched in both human and mouse cells have revealed that levels in mRNA may occur in nuclear speckles [3, 14]. m6A modification occurs in highly conserved regions Furthermore, our studies in zebrafish revealed that MET - with a consensus sequence RRACH (R = G or A; H= A, TL3 mRNA expression level changed during embryonic C or U) [9-13]. The m6A modification occurs in intra - development (also observed in vitro in bovine embryos genic regions particularly in the 3′-end of CDS and the [45]), and that depletion of WTAP and METTL3 com- first quarter of the 3′-UTR [9, 10]. Our PAR-CLIP data promised tissue differentiation (Figure 4 and Supplemen- demonstrate that the conserved WTAP- and METTL3- tary information, Figure S6), strongly suggesting that binding motifs both contain the consensus m6A sequence m6A may play a key role in regulating organismal devel- (Figure 3C), and their binding clusters in CDS and UTR opment. regions significantly overlap with m6A sites (Figure On average, there is 1 m6A modification per 2 000 3E), strongly suggesting a direct connection between the www.cell-research.com | Cell Research npg WTAP as m6A methyltransferase regulatory subunit WMM complex and m6A modification. Interestingly, without normalization of the overall length of the various transcript regions, the majority of WTAP- or METTL3-binding clusters were located in introns (Supplementary information, Figure S4A), which suggests that WMM-dependent m6A modification may be coupled to gene transcription as previously proposed [1-3]. For the relationship of m6A and mRNA splicing, Dominissini and colleagues calculated the average num- ber of m6A peaks in multi-isoform and single-isoform genes, and assigned all m6A peaks to either constitu- Figure 5 A schematic model of the WMM complex function. tively or alternatively spliced exons based on the list of The WMM complex mediates methylation of internal adenosine splicing events of all coding Ensembl genes. Their study residues in eukaryotic mRNA, forming N6-methyladenosine. revealed that m6A modification is significantly over-rep - WTAP binds to the m6A consensus RRACH motif of mRNA and resented in multi-isoform genes and alternatively spliced recruits catalytic subunits METTL3 and METTL14. Then the exons, proposing that m6A may affect RNA splicing [9]. METTL3-METTL14 complex carries out m6A methyltransferase activity in the m6A motif. Consistent with this observation, we found an enrichment of WTAP/METTL3-binding clusters in genes with multi- isoforms and alternatively spliced exon junctions based on our PAR-CLIP+RNA-seq dataset (Figure 3G and 3H). Materials and Methods Taken together, these results suggest that WTAP/MET- TL3 and thereby the m6A methyltransferase may influ - Cell culture, plasmids and antibodies ence RNA splicing. However, a thorough understanding Human cells were cultured in standard cell culture Dulbecco’s of the relationship between m6A and transcription and/or modified Eagle’s medium at 37 °C in a humidified incubator with splicing awaits future investigations. 5% CO (v/v).The human METTL3 gene was cloned into pCMV- Myc (Invitrogen), S-protein/FLAG/SBP (streptavidin-binding Drosophilia female-lethal (2)D (FL (2)D, WTAP protein) triple-tagged destination vector [48] or pGEX-5×-2 (GE homolog) also interacts with the female-specific RNA- healthcare). The human WTAP gene was cloned into p3XFLAG- binding protein sex-lethal (SXL) [46, 47], VIR [46], Snf, CMV-14 (Sigma) or pProEX-HTb (Invitrogen). The human MET- U2AF, U2A50 and U2AF38 [47]. This together with the TL14 gene was cloned into pcDNA3-HA (Invitrogen). The follow- results showing that there is a difference in the numbers ing antibodies were used: rabbit-anti-METTL3 (Abcam), mouse- of genes containing METTL3- or WTAP-binding clus- anti-METTL3 (Abnova), mouse-anti-WTAP (Santa Cruz), rabbit- ters might suggest that METTL3 is the core component anti-METTL14 (Atlas), rabbit-anti-m6A (Synaptic Systems) and other antibodies included in Supplementary information, Data S1. of the m6A methyltransferase, whereas WTAP serves as its regulatory subunit. Based on the findings that WTAP Tandem affinity purification and mass spectrometry interacts directly with METTL3 (Figure 1) and medi- 293T cells transiently transfected with triply-tagged METTTL3 ates nuclear speckle localization of both METTL3 and plasmid (SFB-METTL3) were used for tandem affinity purifica - METTL14, we propose a model, in which METTL3 and tion. Cell lysates were incubated with streptavidin-Sepharose METTL14 are anchored to mRNA by WTAP, allow- beads (Amersham Biosciences), and eluted with 1 mg/ml bio- ing subsequent methylation of target adenosine residues tin (Sigma). The eluates were further incubated with S-protein- agarose (Novagen). The proteins bound to S-protein-agarose beads (Figure 5). were subjected to SDS-PAGE and visualized by coomassie blue In summary, our findings identify WTAP as a regula - staining. The identities of eluted proteins were revealed by mass tory subunit required for formation of a functional m6A spectrometry analysis. The Q Exactive mass spectrometry data methyltransferase complex including METTL3 and (Thermo Fisher Scientific) were searched against SwissProt hu- METTL14, which play an important role in the regula- man database using 15 ppm peptide mass tolerance and 20mmu tion of gene expression and alternative splicing. Thus, fragment mass tolerance. the discovery and characterization of the RNA m6A methyltransferase complex, together with the recent ad- Immunoprecipitation 293T cells transfected with the indicated siRNAs and/or DNA vances in the understanding of the demethylases FTO constructs were lysed in buffer (100 mM NaCl, 20 mM Tris- and ALKBH5, is an important step towards a more thor- HCl (pH 7.4), 0.5% NP-40, 1 mM PMSF, 1 mM Na VO 1 mM 3 4, ough understanding of the biological significance of the β-glycerophosphate, 1 mM NaF and 1× Cocktail), and subjected epitranscriptomic marker m6A. to immunoprecipitation (IP) followed by immunoblotting with the indicated antibodies. Cell Research | Vol 24 No 2 | February 2014 npg Xiao-Li Ping et al . The following antibodies were used in the study: mouse-anti- to the size of Myc-METTL3 or Flag-WTAP. Purified RNAs from Flag (Sigma), rabbit-anti-Myc (Abcam), rabbit-anti-HA(Clontech), RNA-Protein complex were subjected to the small RNA library rabbit-anti-WTAP (Atlas), Anti-Rabbit IgG-HRP (Dakocytoma- construction and then sequenced with HiSeq 2000 system (Illumina tion), Anti-Mouse IgG-HRP (Dakocytomation). Inc.). Bowtie 0.12.7 software [52] was applied to map sequencing reads against human (hg19) genome with up to two mismatches allowed. To multi-mapped reads, mapped locations were only GST pull-down assay The human METTL3 gene was subcloned into pGEX-5X-2 reported for those with the minimum number of observed mis- expression plasmid with GST-tag and the human WTAP gene matches. PARalyzer software [36] was used to define METTL3- was subcloned into pProEX-HTb expression plasmid with His and WTAP-binding sites. Sequence motifs enriched in clusters tag. Recombinant GST-METTL3 and His-WTAP proteins were were identified by HOMER, a suite of tools for motif discovery induced to express in E. coli strain BL21(DE3) and purified by and next-generation sequencing analysis [38]. FPLC using Bio-Scale Mini Profinity GST cartridge (Biorad) and Total RNA was extracted from 293T cells transfected with Bio-Scale Mini Profinity IMAC cartridge (Biorad) as described siRNA using RNAzol (Molecular Research Center, Inc., USA). in the commercial instruction manual. The quality of proteins was cDNA library was constructed using TruSeq RNA Sample Prep tested by western blot and coomassie brilliant blue staining. His- Kit and then sequenced with HiSeq 2000 system (Illumina Inc.). WTAP proteins were incubated with glutathione sepharose (GE RNA-seq reads were aligned against the Ensembl genome (hg19) Healthcare) to be pre-cleared in NETN buffer (100 mM NaCl, 1 using TopHat2 [40, 53]. The number of reads mapped to each of mM EDTA, 20 mM Tris-HCl pH 7.4, 0.5% NP-40). Then pre- the Ensembl genes (version 72) was counted using the HTSeq cleared His-WTAP was mixed with GST or GST-METTL3 protein python package [54]. DEGs were determined using the R-package and incubated overnight at 4 °C with equal amount of glutathione DEGseq with the method MARS (MA-plot-based method with sepharose beads. After washing the beads five times with NETN random sampling model), P value cutoff = 0.001 [55]. GO analy- buffer, proteins bound to the beads were analyzed by 8% SDS- ses of DEGs and PAR-CLIP data were performed using DAVID PAGE followed by immunoblotting with HRP-conjugated anti-His [56, 57]. Enrichment map of DEGs was constructed by Cytoscape and anti-GST antibodies (Abcam). 2.8.3 software [57]. The FPKM (Fragments Per Kilobase of exon per Million mapped reads) value for each transcript was calculated by using Cufflinks version 2.0.0 and the differentially expressed Immunofluorescence staining HeLa cells grown on coverslips were transfected with siRNA transcripts were identified by Cuffdiff program in the Cufflinks (50 nM) or DNA constructs as indicated for about 4 h, and 24 h software package [58]. Scripture, a method for transcriptome re- later fixed with 4% paraformaldehyde on ice followed by permea - construction that relies solely on RNA-Seq reads and an assembled bilization with 0.1% Triton X-100 and 0.05% NP-40 on ice. After genome to build the transcriptome [59], was applied to calculate pre-blocking with 5% non-fat dried milk in TBST, coverslips were the isoform number for each gene. Variation in the isoform number first incubated with primary antibody for 1 h and then with fluo- between METTL3/WTAP-depleted and control samples indicated rescent dye-conjugated secondary antibody for 0.5 h and mounted an alternative splicing event. with DAPI-containing mounting medium (Vector Laboratories, Furthermore, a Bowtie library of non-redundant set of se- Burlingame, CA, USA) and visualized under immunofluorescence quences consisting of all possible junctions between the exons of confocal microscope Leica TCS SP5 (Leica). The method used each Refseq gene of human was generated and all the RNA-seq for immunofluorescence staining following RNase A treatment reads were aligned against this library with Bowtie 0.12.7 software was adapted from Mayer et al. [49] and Sytnikova et al. [50]. In [52]. To the exon-exon junctions (EEJs) with read mapping, ac- brief, cells grown on coverslips were permeabilized with 0.05% cording to their continuous or discrete exon number, they can be Triton X-100 in 20 mM Tris-HCl (pH 7.4), 5 mM MgCl , 0.5 mM divided into constitutively or alternatively spliced exons. All the EDTA, and 25% glycerol; washed twice with PBS; and treated WTAP/METTL3 clusters were assigned to these exons. with RNase A (1 mg/ml) (diluted in PBS) in 37 ºC for 7 min. After washing, cells were fixed with cold methanol (15 min) and stained MO-mediated gene knockdown in zebrafish embryos with the indicated antibodies following the blocking procedure. The antisense MO oligonucleotides targeting individual mem- The immunofluorescence images were digitally recorded by using bers of the WMM complex were synthesized (Gene Tools, LLC.). confocal microscope. The following antibodies were used in im- The MOs (2.5-8 ng) were injected into embryos from one-cell munofluorescence staining: mouse-anti-Flag (Sigma), rabbit-anti- to four-cell stage. Zebrafish WTAP and METTL3 were amplified Myc (Abcam), rabbit-anti-HA (Clontech), mouse-anti-METTL3 from zebrafish cDNA library by PCR and sequentially subcloned (Abnova), rabbit-anti-METTL14 (Atlas), mouse-anti-WTAP (Santa into the pGEM-T vector and pEGFP-N1 (Clontech) to generate Cruz), mouse-anti-SC35 (Sigma), anti-mouse IgG-Cy3 (Sigma), pWTAP-GFP or pMETTL3-GFP constructs for checking MO ef- anti-rabbit IgG-Cy3 (Sigma), anti-mouse IgG-FITC (Sigma), anti- ficiency rabbit IgG-FITC (Sigma). Statistical analysis PAR-CLIP and RNA-Seq Two-tailed t-tests in grouped analyses of Prism5 software 293T cells expressing Myc-METTL3 or Flag-WTAP were cul- (GraphPad Software Inc., USA) were applied. P-values < 0.05 tured in medium supplemented with 4-SU (Sigma), irradiated with were considered significant. 365 nm UV light to induce crosslinking as described previously [51]. Immunoprecipitated protein-RNA complexes were separated Accession numbers by SDS-PAGE and then transferred to PVDF membrane; RNA- Mass spectrometry data have been uploaded to Peptide Atlas protein complexes were cut out from the membrane corresponding under accession number PASS00296. PAR-CLIP and RNA-Seq www.cell-research.com | Cell Research npg WTAP as m6A methyltransferase regulatory subunit data have been uploaded to GEO database and can be accessed via adenosine sites of HeLa cell messenger ribonucleic acid. Bio- accession number GSE50583. chemistry 1977; 16:1672-1676. 14 Jia G, Fu Y, Zhao X, et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Acknowledgments Chem Biol 2011; 7:885-887. 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Temporal expression of factors involved in chromatin remodeling and in gene regula- This work is licensed under the Creative Commons tion during early bovine in vitro embryo development. Repro- Attribution-NonCommercial-No Derivative Works duction 2007; 133:597-608. 3.0 Unported License. To view a copy of this license, visit http:// 46 Ortega A, Niksic M, Bachi A, et al. Biochemical function creativecommons.org/licenses/by-nc-nd/3.0 www.cell-research.com | Cell Research

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