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Alternative splicing of METTL3 explains apparently METTL3-independent m6A modifications in mRNA [1]

['Hui Xian Poh', 'Department Of Pharmacology', 'Weill Cornell Medicine', 'Cornell University', 'New York', 'United States Of America', 'Aashiq H. Mirza', 'Brian F. Pickering', 'Samie R. Jaffrey']

Date: 2022-07

N 6 -methyladenosine (m 6 A) is a highly prevalent mRNA modification that promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m 6 A in mRNA. As 30% to 80% of m 6 A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m 6 A. Here, we reexamined the source of m 6 A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines that continue to have m 6 A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3’s role in synthesizing m 6 A using an exon 4 deletion of Mettl3 and found that METTL3 is responsible for >95% of m 6 A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m 6 A in the transcriptome, and that remaining m 6 A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms.

Funding: This work was supported by National Institutes of Health (NIH, https://www.nih.gov/ ) grants R35NS111631, R01CA186702, and S10OD030335 to S.R.J and F32CA22104 to B.F.P., and by Agency for Science, Technology And Research (A*STAR, https://www.a-star.edu.sg/ ) National Science Scholarship to H.X.P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we address the source of m 6 A in mRNA in reported METTL3 knockout cells. We examined two different Mettl3 knockout mESC lines, which both report loss of METTL3, but showed vastly different levels of residual m 6 A in mRNA. We show that Mettl3 mutagenesis by CRISPR approaches can create alternatively spliced isoforms of Mettl3, resulting in an altered but catalytically active METTL3 protein. Thus, the residual m 6 A can be ascribed to a hypomorphic METTL3 allele. We further show that other published METTL3 mutant cell lines, which were intended to delete METTL3, retain m 6 A and express alternative METTL3 proteins. Furthermore, we show that METTL3 is an essential gene in most cell lines, and thus, METTL3 knockout cell lines that remain viable are likely to have generated alternatively spliced functional METTL3 proteins that bypass the CRISPR mutations. Lastly, we show that when a large deletion is created in METTL3, essentially all m 6 A is depleted in a fibroblast cell line. Overall, these studies argue that METTL3 is responsible for most m 6 A in mRNA, and that residual m 6 A after METTL3 depletion usually reflects the generation of hypomorphic METTL3 alleles and therefore incomplete METTL3 knockout.

METTL3 has been knocked out in other cell lines and tissues. These results have shown that 30% to 80% of m 6 A can remain after METTL3 knockout [ 23 – 33 ]. In U2OS osteosarcoma cells, approximately 60% of m 6 A remained after CRISPR-mediated knockout of METTL3 [ 23 , 24 ]. In HEK293T human embryonic kidney cells, approximately 50% of m 6 A remained after CRISPR-mediated knockout of METTL3 [ 25 ]. After Cre-conditional genomic deletion of Mettl3 in mouse CD4+ T cells, 28% of m 6 A remained [ 30 ]. Since a nonnegligible amount of m 6 A persists after METTL3 knockout, it has been speculated that other methyltransferases may have a major role in forming m 6 A in mRNA [ 4 , 31 , 33 – 35 ].

Although METTL3 is often described as the major m 6 A-forming enzyme in cells, the amount of m 6 A thought to be formed by METTL3 varies widely in different studies. The first study to knockout Mettl3 showed that approximately 40% of m 6 A remained after Mettl3 knockout in mouse embryonic stem cells (mESCs) [ 4 ]. The authors suggested that METTL14 may account for this 40% of m 6 A, based on the previous understanding that METTL14 was catalytic. However, a different group shortly thereafter reported that deletion of either Mettl3 or Mettl14 in mESCs leads to a loss of approximately 99% of m 6 A in mRNA [ 5 ]. These contradictory results have led to uncertainty about how much m 6 A in mRNA derives from METTL3.

The first enzyme shown to catalyze m 6 A formation was METTL3 [ 18 ], which forms a heterodimer complex with METTL14 [ 19 – 21 ]. METTL3 contains the catalytic component. METTL14 was initially believed to have catalytic ability [ 21 ], but METTL14 is now known to be catalytically inactive [ 19 , 20 ]. Instead, METTL14 binds and positions RNA for methylation [ 19 , 20 ]. The METTL3-METTL14 complex is a component of a larger multiprotein “m 6 A writer complex” that mediates co-transcriptional mRNA methylation [ 17 , 22 ].

Results

Two mESC lines exhibit different levels of m6A after Mettl3 knockout To understand how much m6A in mRNA is catalyzed by METTL3, we examined 2 previously reported Mettl3 knockout mESC lines. Two groups independently knocked out Mettl3 in mESCs and reported markedly different levels of residual m6A levels in mRNA [4,5]. The first Mettl3 knockout mESC line was described by Batista and colleagues from Howard Chang’s group, and used a CRISPR/Cas9 approach [4]. The guide RNAs were designed to introduce deletions in exon 2 of Mettl3 and create premature termination codons [4]. The resulting mESC line, designated “exon2 Mettl3 KO mESCs,” was found to have 40% residual m6A in mRNA. These authors understandably attributed the remaining m6A to METTL14 since, at that time, METTL14 was incorrectly shown to be a functional methyltransferase [21]. The second mESC line was described by Geula and colleagues from Jacob Hanna’s group [5]. This group used loxP sites surrounding exon 4 in Mettl3 to delete the exon encoding the zinc finger domain (ZFD), an RNA recognition domain required for METTL3 methyltransferase activity [19,36]. This Mettl3 knockout mESC line, designated “exon4 Mettl3 KO mESCs,” exhibited <1% remaining m6A in mRNA. It is unclear why the exon2 Mettl3 KO mESCs have high m6A levels when the exon4 Mettl3 KO mESCs, which in principle should be the same, have virtually no remaining m6A. We first reconfirmed the levels of m6A in these two cell lines. The m6A levels in the two Mettl3 knockout mESC lines were originally measured via different methods, which may have led to these contradictory results. Thin-layer chromatography was used by Batista and colleagues [4], while mass spectrometry was used by Geula and colleagues [5]. We measured m6A in the mESC lines using mass spectrometry [37]. Our mass spectrometry measurements were consistent with the results originally reported by the two groups. In the two exon2 Mettl3 KO mESC lines, designated exon2 Mettl3 KO mESC-a and exon2 Mettl3 KO mESC-b, we saw 40.2% and 55.6% residual m6A (Fig 1A), comparable to approximately 40% originally reported by this group [4]. However, the exon4 Mettl3 KO mESCs had only 1.45% residual m6A (Fig 1A), corroborating the 0.5% remaining m6A originally reported by this group [5]. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Previously described Mettl3 KO mESC lines express shorter isoforms of Mettl3. (A) Mettl3 KO mESCs from two groups have different m6A levels. To reconfirm the m6A levels using quantitative methods, we performed mass spectrometry to estimate the m6A in mRNA. Exon2 Mettl3 KO mESCs show persistence of 40.2% (exon2 Mettl3 KO mESC-a) and 55.6% (exon2 Mettl3 KO mESC-b) m6A, respectively, while exon4 Mettl3 KO mESCs show 1.45% m6A compared to WT. This confirms that exon4 Mettl3 KO mESCs have near-complete loss of m6A, but not the exon2 Mettl3 KO mESCs. Error bars indicate standard error (n = 3 for all, except n = 2 for exon2 Mettl3 KO mESC-b). * = p-value < 0.5, ** = p-value < 0.01, *** = p-value < 0.005, n.s. = not significant. Underlying data can be found in S1 Data. (B) Exon2 Mettl3 KO mESCs exhibit new anti-METTL3-immunoreactive bands. To investigate the effectiveness of the Mettl3 knockout, we measured the loss of METTL3 via WB. Full-length METTL3 (75 kDa, arrowhead) was lost in both KO cell lines, but new bands, which were reactive to the anti-METTL3-antibody, appeared at approximately 50 kDa in exon2 Mettl3 KO mESC-a and approximately 55 kDa in exon2 Mettl3 KO mESC-b (arrowheads). This indicates the possibility that a novel smaller METTL3 protein was expressed in the exon2 Mettl3 KO mESCs. In contrast, exon4 Mettl3 KO mESCs have no proteins reactive to anti-METTL3-antibodies. 30 μg per lane. (C) 5′ RACE reveals the expression of shorter Mettl3 mRNAs in the Mettl3 KO mESCs. We used 5′ RACE to identify novel Mettl3 mRNAs in the Mettl3 KO mESCs. The full-length RACE product (approximately 1,500 bp) was lost in the Mettl3 KO cells, but novel products at approximately 1,000 bp and approximately 700 bp were found in exon2 Mettl3 KO mESC-a and at approximately 1,500 bp and approximately 1,300 bp in exon2 Mettl3 KO mESC-b. These shorter mRNAs may encode the smaller METTL3 proteins seen in the KO cells. (D) Sequencing of 5′ RACE products show Mettl3 mRNAs with exon skipping or alternative transcription-start sites. We sequenced the 5′ RACE products to characterize the Mettl3 mRNA transcripts that are expressed by the exon2 Mettl3 KO mESCs. All Mettl3 mRNAs expressed in the KO cells skipped the guide RNA deletion region by exon skipping, or by using alternative transcription-start sites downstream of the deletion. The longest ORFs that are in-frame with the WT Mettl3 mRNAs are shown as solid lines below each mRNA. The encoded protein is also represented, with the domains required for METTL3 activity shown. m6A, N6-methyladenosine; mESC, mouse embryonic stem cell; NLS, nuclear localization signal; ORF, open reading frame; pAb, polyclonal antibody; RACE, rapid amplification of cDNA ends; WB, western blot; WT, wild-type; ZFD, zinc finger domain. https://doi.org/10.1371/journal.pbio.3001683.g001 The near-complete loss of m6A in the exon4 Mettl3 KO mESCs suggests that METTL3 is the major m6A writer in this mESC line. On the other hand, the exon2 Mettl3 KO mESCs still retain m6A despite Mettl3 depletion. Although it is possible that these mESCs use an alternate enzyme for m6A biosynthesis, we suspected that Mettl3 was not completely knocked out in these cell lines.

Mettl3 knockout mESCs that retain m6A express alternative Mettl3 isoforms We next wanted to confirm that Mettl3 was knocked out in the exon2 Mettl3 KO mESCs. Previously, a western blot was used to determine the loss of METTL3 protein [4]. To first confirm that the METTL3 protein is indeed absent, we performed a western blot using a METTL3 polyclonal antibody raised against amino acids 229–580 of METTL3, which correspond to amino acids encoded by exons 3–11. Full-length METTL3 (approximately 75 kDa) was identified in the wild-type (WT) mESCs, and was lost in both Mettl3 KO cell lines (Fig 1B). However, we observed new bands in the anti-METTL3 immunoblot in the knockout cell lines. The new proteins were approximately 50 kDa in exon2 Mettl3 KO mESC-a and approximately 55 kDa in exon2 Mettl3 KO mESC-b (Fig 1B). While nonspecific background bands are visible in all 3 cell lines, these particular proteins were not visible in the WT cell line, suggesting they are unique to the knockout cell lines and not just background. These proteins were also not visible in the exon4 Mettl3 KO cell lines (Figs 1B and S1B). To confirm that these are indeed METTL3 proteins, we repeated the western blot with a second anti-METTL3 antibody raised against amino acids surrounding Leu297 of METTL3, which correspond to amino acids encoded by exon 4. Again, we found the same bands in the exon2 Mettl3 KO cell lines (S1A and S1C Fig). These proteins may have escaped notice in the original study as the study used a different antibody, which may have been unable to detect these isoforms [4]. Overall, the new METTL3-antibody-reactive proteins suggests that smaller METTL3 isoforms are produced in the exon2 Mettl3 KO cells that may be the source of m6A in these cells. Although expression levels of these proteins appear low, previous studies have suggested that METTL3 is not rate limiting, and therefore, low METTL3 expression can still lead to high m6A levels [22]. We wanted to understand the mechanism of METTL3 expression in the exon2 Mettl3 KO mESCs. To do this, we first determined the sequence of these isoforms. Since the exon2 Mettl3 KO cells were produced using guide RNAs targeting exon 2 of Mettl3 [4], any CRISPR/Cas9-induced mutations and potential alternative splicing events are likely to be in the 5′ end of the transcript. We therefore used 5′ RACE (rapid amplification of cDNA ends) [38] to identify new transcription-start sites or possible exon skipping events of the Mettl3 transcripts expressed in these cells. Using 5′ RACE, a single major approximately 1,500 bp band was seen for Mettl3 in WT mESCs (Fig 1C). In contrast, in the two exon2 Mettl3 KO cell lines, we found shorter bands indicative of Mettl3 mRNAs with a shorter 5′ region (Fig 1C). We sequenced these 5′ RACE products and found several Mettl3 mRNAs from the exon2 Mettl3 KO cells (Fig 1D and S1 Table). In all cases, the mRNAs show alternative splicing that bypasses the CRISPR deletion in exon 2 by exon skipping, or use of an alternative transcription-start site downstream of the deletion. We next asked if these mRNAs could potentially encode the altered METTL3 proteins detected in the Mettl3 KO mESCs. For each mRNA, we identified the longest possible open reading frame (ORF) that is in frame with the METTL3 catalytic domain (Fig 1D and S2 Table). In the case of Mettl3 KO mESC-a, we found a transcript that is predicted to encode a METTL3 protein (designated “METTL3-a.ii”) that matches the size of the altered METTL3 protein from this cell line (Fig 1B and 1D). Similarly, we identified a transcript that is predicted to encode a protein with a similar size to the altered METTL3 protein in Mettl3 KO mESC-b (designated “METTL3-b.ii”) (Fig 1B and 1D). Together, these data identify potential transcripts that may encode the altered METTL3 proteins found in the exon2 Mettl3 KO cells.

METTL3 knockout U2OS cells express an altered METTL3 protein The idea that METTL3 is not responsible for all m6A in mRNA was further supported by METTL3 knockout in several cell lines, each of which show high residual levels of m6A [23–29]. For example, a METTL3 KO U2OS cell line was reported to have approximately 60% of the m6A remaining compared to WT [23,24]. Similarly, we found that a previously reported METTL3 KO A549 cell line [41] has approximately 90% of m6A remaining compared to the WT (S5 Fig). The persistence of m6A in these METTL3 knockout cell lines has led to the idea that other enzymes mediate a substantial fraction of m6A in mRNA. However, in light of our finding that alternatively spliced Mettl3 isoforms were induced in the Mettl3 knockout mESCs, another possibility is that METTL3 isoforms were induced in these METTL3 knockout cell lines as well, which led to the high m6A levels in these METTL3 knockout cell lines. We examined one of these cell lines—the METTL3 knockout U2OS cell line that was generated using CRISPR/Cas9-directed mutagenesis with a guide RNA targeting exon 1 [23]. We first reconfirmed the m6A levels in these METTL3 knockout cells and found that the METTL3 knockout U2OS cells retained 75.2% m6A compared to the WT (Fig 3A), comparable to the 60% originally reported [24]. Hence, METTL3 knockout U2OS cells continue to have high levels of m6A in mRNA. PPT PowerPoint slide

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TIFF original image Download: Fig 3. METTL3 knockout in U2OS cells also appears to be incomplete. (A) METTL3 KO U2OS cells have persistent m6A. METTL3 KO U2OS cells have been reported to have 60% the levels of m6A found in control U2OS cells [24]. We reconfirmed this with mass spectrometry measurements of m6A, which showed that METTL3 KO U2OS cells have 75.2% remaining m6A compared to WT. Thus, m6A levels remain high in METTL3 KO U2OS cells. Error bars indicate standard error (n = 3). * = p-value < 0.5, ** = p-value < 0.01, *** = p-value < 0.005, n.s. = not significant. Underlying data can be found in S1 Data. (B) METTL3 KO U2OS cells express a novel anti-METTL3-antibody-reactive protein. As m6A levels were not completely ablated in the METTL3 KO U2OS cells, we assessed METTL3 protein expression in these cells to confirm if the knockout was effective. We found that WT METTL3 protein was lost in the METTL3 KO U2OS cells, but a larger protein that was reactive to the anti-METTL3 antibody was found in the METTL3 KO U2OS cells. This suggests the METTL3 KO U2OS cells express a novel METTL3 protein that is slightly larger than the WT METTL3. (C) Confirmation of a novel METTL3-like protein in METTL3 KO U2OS using a second METTL3 antibody. To confirm that the METTL3-immunoreactive band we saw in METTL3 KO U2OS cells in Fig 3B was METTL3, we used a second anti-METTL3 mAb to confirm the result. The same protein band is immunoreactive to the second anti-METTL3 antibody, thus suggesting that the METTL3 KO U2OS cells express a novel, larger METTL3 protein. (D) A METTL3-specific inhibitor leads to loss of m6A even in METTL3 KO U2OS cells. WT and METTL3 KO U2OS cells were treated with 30 μM STM2457, and m6A levels were measured by mass spectrometry after 48 h. m6A was reduced by 89.8% in the WT U2OS cells and 92.1% in the METTL3 KO U2OS cells after STM2457 treatment. It should be noted that 30 μM may not fully inhibit METTL3, so some of the residual m6A after STM2457 treatment may still derive from METTL3 isoforms. Thus, a METTL3 isoform is responsible for most of the remaining m6A in the METTL3 KO U2OS cells. Error bars indicate standard error (n = 3). * = p-value < 0.5, ** = p-value < 0.01, *** = p-value < 0.005, n.s. = not significant. Underlying data can be found in S1 Data. mAb, monoclonal antibody; m6A, N6-methyladenosine; pAb, polyclonal antibody; WB, western blot; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001683.g003 We next asked if the METTL3 KO U2OS cells express a METTL3 protein using a western blot. We observed full-length METTL3 in the WT U2OS cells, but not in the knockout cells (Fig 3B). However, a slightly larger anti-METTL3 immunoreactive band was detected exclusively in the knockout cells (Fig 3B). To confirm that this is indeed a METTL3 isoform, we validated the western blot with a second anti-METTL3 monoclonal antibody and found that the protein in the knockout cells was also reactive to the second anti-METTL3 antibody (Fig 3C). We note that we could observe a similar protein band in the authors’ original western blot that used a different METTL3 antibody [23]; however, it was much fainter and could easily be mistaken for nonspecific background. Thus, this suggests that the METTL3 KO U2OS cells continued to express a METTL3 protein. We wanted to find out if METTL3 could be responsible for the m6A produced in the METTL3 KO U2OS cells using the METTL3-specific inhibitor, STM2457 [40]. STM2457 treatment reduced m6A levels in the WT U2OS cells by 89.8% after 48 h (Fig 3D). Similarly, STM2457 treatment reduced m6A levels by 92.1% in the METTL3 KO U2OS cells (Fig 3D). These data suggest that a METTL3 isoform is responsible for most of the remaining m6A in the METTL3 KO U2OS cells. Furthermore, the depletion of approximately 90% m6A by STM2457 further suggests that METTL3 is responsible for the majority of m6A in U2OS cells. Overall, this data again suggests that CRISPR/Cas9 mutagenesis results in the appearance of a novel METTL3 isoform in a METTL3 knockout cell line, which could explain m6A persistence in these cells.

METTL3 knockout cell lines are generally not viable Several METTL3 knockout cell lines have been reported despite the fact that METTL3 is thought to be an essential gene. Mettl3 knockout is embryonic lethal in mice at E5.5 before cell specification occurs [5], and CRISPR screens have indicated that METTL3 is an essential gene in specific cell lines that were tested [7,42]. On the other hand, the exon4 Mettl3 knockout mESCs are able to survive, demonstrating that some cell lines can survive without METTL3 or m6A. Therefore, it is not clear which cell lines require METTL3 for survival. If METTL3 is required for survival of most cell lines, it would cast doubt on the stable METTL3 knockout cell lines that have been reported in the literature. To understand which cell lines require METTL3 for survival, we screened the Cancer Dependency Map Project (DepMap) 21Q4 dataset [43–47]. This dataset measures cell proliferation in 1,054 cell lines following a CRISPR loss-of-function screen. Failure of a cell line to grow after expression of the guide RNA that inactivates a gene indicates that the gene is essential in the cell line. The probability that a cell line is dependent on each gene is calculated as a gene dependency probability score, which accounts for guide efficacy as well as copy number of each gene. The 21Q4 DepMap dataset [47] showed that METTL3 is necessary for cell proliferation in 801 of 1,054 tested cell lines (Fig 4A). To account for the possibility of off-target effects in the DepMap CRISPR screen, we further looked for METTL3-dependent cell lines that were also dependent on other members of the writer complex, METTL14 and WTAP [6,21,22,48]. We found that of the 801 cell lines dependent on METTL3, 683 were also dependent on METTL14 and WTAP (S6 Fig), suggesting that these cell lines indeed require the m6A methyltransferase complex for proliferation. The METTL3-dependent cell lines include the U2OS and A549 cell lines that other groups have used to make METTL3 knockout cell lines [23,41] (Fig 4A). The DepMap data suggests that these reported METTL3 cell lines should not have been viable. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Conditional METTL3 knockouts can be used to study m6A when stable METTL3 knockouts are not viable. (A) Most cell lines are dependent on METTL3 for growth. Mouse studies previously indicated that Mettl3 is an essential gene for early embryonic survival [5], so we wanted to know which cell lines are dependent on METTL3. Using the CRISPR gene dependency probability score from the DepMap 21Q4 dataset [43–47], we found that 801 of 1,054 cell lines are dependent on METTL3 (dependency probability score >0.5). Therefore, most cells lines will not survive after METTL3 knockout. The density plot shows the overall distribution of dependency probability scores, while each individual cell line is represented as a dot. U2OS and A549 cell lines (red), where METTL3 has been previously knocked out, are shown here to be dependent on METTL3 and thus should not be viable after METTL3 knockout. Underlying data for this figure was extracted from the DepMap 21Q4 dataset [47]. (B) A small subset of cell lines may be m6A independent. Although most cell lines are dependent on METTL3, a small subset of cell lines can survive despite METTL3 knockout. To identify cell lines that we can confidently consider m6A-independent, we obtained a list of cell lines whose survival is also independent of other members of the m6A writer complex, METTL3, METTL14, and WTAP. This approach suggests that 65 cell lines may be able to proliferate in an m6A-independent manner (S4 Table). Underlying data for this figure was extracted from the DepMap 21Q4 dataset [47]. (C) Mettl3 conditional knockout MEFs do not express METTL3 protein. To determine the amount of m6A in mRNA that can be attributed to Mettl3, we generated a tamoxifen-inducible Mettl3 conditional knockout MEF cell line. We used WB to validate the loss of METTL3. After 5 days of 4-hydroxytamoxifen treatment (500 nM), we observed loss of the WT METTL3 protein. 30 μg per lane. (D) Mettl3 conditional knockout MEFs show near-complete loss of m6A. We measured m6A levels in mRNA derived from tamoxifen-inducible Mettl3 conditional knockout MEFs. Eight days after 4OHT treatment (500 nM), the Mettl3 KO MEFs showed 3.6% remaining m6A. Hence, m6A is almost completely lost after Mettl3 knockout in MEFs. Error bars indicate standard error (n = 3). * = p-value < 0.5, ** = p-value < 0.01, *** = p-value < 0.005, n.s. = not significant. Underlying data can be found in S1 Data. m6A, N6-methyladenosine; MEF, mouse embryonic fibroblast; pAb, polyclonal antibody; WB, western blot; 4OHT, 4-hydroxytamoxifen. https://doi.org/10.1371/journal.pbio.3001683.g004 It is important to note that the DepMap dataset measures proliferation after a CRISPR loss-of-function screen. Even if a small number of cells manage to escape the CRISPR inactivation of METTL3 and continue to proliferate, the overall reduction in proliferation will still be reflected in the DepMap gene dependency score. On the other hand, during generation of METTL3 knockout cell lines, researchers are actively selecting for cells that continue to proliferate, therefore potentially selecting for cells that have escaped the knockout. Thus, this leads us to believe that reported stable METTL3 knockout cell lines were able to survive because they were selected for their ability to produce a functional alternatively spliced METTL3 isoform that bypasses the CRISPR mutation, similar to the exon2 Mettl3 KO mESCs. This alternative METTL3 isoform also explains why these cells retain high levels of m6A. Thus, the reported METTL3 knockout cell lines are likely not true METTL3 knockouts. However, a few cell lines are able to survive after METTL3 knockout. The exon4 Mettl3 KO mESCs are one such example of a cell that can survive without METTL3 [5]. A group has also reported that mouse CD4+ T cells can survive after Mettl3 deletion [30]. The 21Q4 DepMap dataset shows that only 253 cell lines are not dependent on METTL3. Furthermore, only 65 cell lines were not dependent on any of the members of the m6A writer complex, which includes METTL3, METTL14, and WTAP [6,21,22,48] (Fig 4B and S4 Table). This small subset of cell lines may be the best cell lines for creating METTL3 knockout cells, since loss of m6A in the other cell lines will most likely lead to cell death unless an alternatively spliced METTL3 isoform is selected for. Thus, any reported stable METTL3 knockout in cell lines other this small subset of m6A-independent cell lines should be more carefully evaluated.

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