FTO stabilizes MIS12 and counteracts senescence

N-methyladenosine (mA) is an abundant epitranscriptomic modification that regulates messenger RNA (mRNA) biology. The mA modification regulates mRNA splicing, transport, stability, and translation through coordinated activities by methyltransferases (writers), binding proteins (readers), and demethylases (erasers) (Huang et al., 2020; Wu et al., 2020). Among mA regulators, fat mass of obesity-associated protein (FTO), is the first discovered eraser with RNA mA demethylation activity (Jia et al., 2011). Since then, FTO has been reported to play mA-dependent roles in a variety of physiological processes including adipogenesis, neurogenesis and tumorigenesis (Fischer et al., 2009; Li et al., 2017; Huang et al., 2020). Consequently, FTO deficiency in mice leads to dramatic phenotypes, such as decreased fat mass and impaired brain development (Fischer et al., 2009; Li et al., 2017). Similarly, inhibition of FTO reduces tumorigenesis in multiple types of cancer models, while FTO is highly expressed in many cancers (Huang et al., 2020). Although FTO is primarily linked with biological functions related to demethylation of internal mA, recent work indicates a more complex role in FTO-mediated epitranscriptional regulation. Specifically, FTO was reported to demethylate other substrates, such as N,2′-O-dimethyladenosine (mAm) and N -methyladenosine (mA), and, based on its varied cellular distribution across cell types, proposed to be afforded differential access to RNA substrates (Wei et al., 2018; Sun et al., 2021). Indeed, in recent work, we discovered that mA RNA methylation is reduced in human stem cell models of senescence, and that mA-dependent mRNA stabilization of MIS12, a kinetochore component, protects against senescence (Wu et al., 2020). However, whether and how FTO regulates human stem cell homeostasis remain largely unexplored. Here, using CRISPR/Cas9-based strategy, we generated FTO-deficient human stem cell models and demonstrated an mA-independent way by which FTO stabilizes MIS12 and antagonizes human stem cell senescence. To investigate the role of FTO in regulating human stem cell homeostasis, we first generated FTO-deficient human embryonic stem cells (hESCs) through CRISPR/Cas9-based gene editing (Fig. 1A and 1B). FTO depletion was verified by western blotting and immunofluorescence staining (Figs. 1C, 1D and S1A). Furthermore, we did not detect any off-target effects resulting from FTO deletion (Fig. S1B). Although a previous study reported potential large structural variations near the target site induced by CRISPR-editing in hESCs (Bi et al., 2020), karyotype and copy number variation analyses revealed that the genomic integrity of FTO-deficient (FTO) hESCs was well maintained (Fig. S1C–F). Relative to wild type (WT, FTO) hESCs, DNA hypomethylation at the OCT4 promoter region and the expression of typical pluripotency markers, including OCT4, SOX2 and NANOG, were comparable in FTO hESCs (Figs. 1E, S1G and S1H). Immunofluorescence staining and flow cytometry analysis further showed comparable Ki67-positive cell numbers and cell cycle status between FTO and FTO hESCs (Fig. 1F and 1G). Similarly, we did not detect any remarkable differences in the expression of nuclear laminaassociated protein LAP2 and heterochromatin relevant proteins HP1α and H3K9me3, whose downregulation was associated with stem cell senescence (Shan et al., 2021; Li et al., 2022), between FTO and FTO hESCs (Figs. 1H and S1I–K). Altogether, these data indicate that FTO is dispensable for maintaining hESC self-renewal and the expression of pluripotency genes. Next, we differentiated FTO and FTO hESCs into human mesenchymal progenitor cells (hMPCs) for addressing the effects of FTO deficiency on human adult stem cells (Fig. 1A). Both FTO and FTO hMPCs expressed typical hMPC surface markers including CD73, CD90, and CD105 (Fig. S1L). We confirmed the loss of FTO protein by western blotting and immunofluorescence staining (Figs. 1I, 1J and S1M). Compared with FTO hMPCs, FTO hMPCs were able to differentiate into adipocytes, chondrocytes, and osteoblasts, although with a differentiation bias towards the osteoblast fate at the expense of differentiation towards the adipocyte fate (Fig. S1N–P). Strikingly, and different from what we observed in hESCs, self-renewal capacity was impaired in FTO hMPCs, as indicated by early-onset growth arrest, restricted clonal expansion, decreased Ki67-positive cells, reduced S phase and prolonged G0/G1 phase (Fig. 1K–N). In addition, FTO ablation led to accelerated senescence, as manifested by elevated senescence-associated β-galactosidase (SA-β-gal)

N 6 -methyladenosine (m 6 A) is an abundant epitranscriptomic modification that regulates messenger RNA (mRNA) biology. The m 6 A modification regulates mRNA splicing, transport, stability, and translation through coordinated activities by methyltransferases (writers), binding proteins (readers), and demethylases (erasers) Wu et al., 2020). Among m 6 A regulators, fat mass of obesity-associated protein (FTO), is the first discovered eraser with RNA m 6 A demethylation activity (Jia et al., 2011). Since then, FTO has been reported to play m 6 A-dependent roles in a variety of physiological processes including adipogenesis, neurogenesis and tumorigenesis (Fischer et al., 2009;Li et al., 2017;. Consequently, FTO deficiency in mice leads to dramatic phenotypes, such as decreased fat mass and impaired brain development (Fischer et al., 2009;Li et al., 2017). Similarly, inhibition of FTO reduces tumorigenesis in multiple types of cancer models, while FTO is highly expressed in many cancers .
Although FTO is primarily linked with biological functions related to demethylation of internal m 6 A, recent work indicates a more complex role in FTO-mediated epitranscriptional regulation. Specifically, FTO was reported to demethylate other substrates, such as N 6 ,2′-O-dimethyladenosine (m 6 A m ) and N 1 -methyladenosine (m 1 A), and, based on its varied cellular distribution across cell types, proposed to be afforded differential access to RNA substrates (Wei et al., 2018;Sun et al., 2021). Indeed, in recent work, we discovered that m 6 A RNA methylation is reduced in human stem cell models of senescence, and that m 6 A-dependent mRNA stabilization of MIS12, a kinetochore component, protects against senescence (Wu et al., 2020). However, whether and how FTO regulates human stem cell homeostasis remain largely unexplored. Here, using CRISPR/Cas9-based strategy, we generated FTO-deficient human stem cell models and demonstrated an m 6 A-independent way by which FTO stabilizes MIS12 and antagonizes human stem cell senescence.
To investigate the role of FTO in regulating human stem cell homeostasis, we first generated FTO-deficient human embryonic stem cells (hESCs) through CRISPR/Cas9-based gene editing ( Fig. 1A and 1B). FTO depletion was verified by western blotting and immunofluorescence staining (Figs. 1C, 1D and S1A). Furthermore, we did not detect any off-target effects resulting from FTO deletion (Fig. S1B). Although a previous study reported potential large structural variations near the target site induced by CRISPR-editing in hESCs (Bi et al., 2020), karyotype and copy number variation analyses revealed that the genomic integrity of FTO-deficient (FTO −/− ) hESCs was well maintained (Fig. S1C-F). Relative to wild type (WT, FTO +/+ ) hESCs, DNA hypomethylation at the OCT4 promoter region and the expression of typical pluripotency markers, including OCT4, SOX2 and NANOG, were comparable in FTO −/− hESCs (Figs. 1E, S1G and S1H). Immunofluorescence staining and flow cytometry analysis further showed comparable Ki67-positive cell numbers and cell cycle status between FTO +/+ and FTO −/− hESCs ( Fig. 1F and 1G). Similarly, we did not detect any remarkable differences in the expression of nuclear laminaassociated protein LAP2 and heterochromatin relevant proteins HP1α and H3K9me3, whose downregulation was associated with stem cell senescence Li et al., 2022), between FTO +/+ and FTO −/− hESCs (Figs. 1H and S1I-K). Altogether, these data indicate that FTO is dispensable for maintaining hESC self-renewal and the expression of pluripotency genes.
We then asked whether the accelerated senescence of FTO −/− hMPCs is caused by alterations in the substrate abundance of FTO. Through dot blotting and liquid chromatography assays coupled with tandem mass spectrometry (LC-MS/MS) analysis, we found comparable m 6 A levels in both total RNA and mRNA between FTO +/+ and FTO −/− hESCs ( Fig. S2A-C), and between FTO +/+ and FTO −/− hMPCs ( Fig. S2D-F). Meanwhile, m 6 A abundance, as detected by fluorometric assays, was also similar between FTO +/+ and FTO −/− hESCs or hMPCs ( Fig. S2G and S2H). We then went on to conduct m 6 A-methylated RNA immunoprecipitation sequencing (MeRIP-seq) ( Fig. S2I and S2J), and identified similar motif enrichment and distribution patterns of m 6 A methylation between FTO +/+ and FTO −/− hESCs or hMPCs ( Fig. 2A-D). There was also no increase in the whole-transcriptome m 6 A peak intensity in FTO-deficient hESCs or hMPCs ( Fig. S2K and S2L). Finally, LC-MS/ MS analysis of the m 6 A m modification in mRNA and dot blot analysis of the m 1 A modification in transfer RNA (tRNA) revealed no statistical difference between FTO +/+ and FTO −/− hESCs, and between FTO +/+ and FTO −/− hMPCs ( Fig. S2M-P). In all, these data demonstrate that FTO depletion exerts minimal impact on global m 6 A, m 6 A m and m 1 A levels in both hESCs and hMPCs, suggesting that FTO may regulate the homeostasis of hMPCs in a demethylation-independent manner.
Previous studies reported that FTO interacts with downstream proteins to enhance their biological activity (Tao et al., 2020). To identify such a potential mechanism for FTO in regulating hMPC senescence, we ectopically expressed FLAG-tagged FTO proteins in HEK293T cells and conducted co-immunoprecipitation (co-IP) assay with an anti-FLAG antibody, followed by LC-MS/MS analysis ( Fig. 2E and Table S1). We identified a panel of potential FTO-interacting proteins (Table S1) that were found to be mostly enriched for "mitotic cell cycle process" through Gene Ontology (GO) enrichment analysis (Fig. 2F). Among these was MIS12 ( Fig. S2Q and S2R), a key regulator of cell cycle distribution that we had reported in the regulation of hMPC senescence (Wu et al., 2020). Here, we validated the interaction between FTO and MIS12 through co-IP assay (Fig. 2G). Then, we

Protein & Cell
FTO stabilizes MIS12 and counteracts senescence LETTER asked whether MIS12 protein levels might be influenced by FTO depletion. Indeed, western blot analysis revealed a marked decrease of MIS12 expression in FTO −/− hMPCs rather than in FTO −/− hESCs (Figs. 2H and S2S), indicating a cell-type-specific regulation. When we analyzed the mRNA and m 6 A levels of MIS12, we found no remarkable differences between FTO +/+ and FTO −/− hMPCs, as revealed by the MeRIP-seq and RT-qPCR results ( Fig. S2T-W). These data indicate that FTO regulates the expression of MIS12 protein in an m 6 A-independent manner. We hence speculated that FTO might regulate hMPC senescence by stabilizing MIS12 protein. Indeed, in protein stability assay, we detected an increased MIS12 degradation upon FTO deficiency (Fig. 2I). To confirm the pathway by which FTO ablation facilitated the protein degradation of MIS12, we treated FTO −/− hMPCs with MG132 (a proteasome inhibitor) and bafilomycin A1 (BFA1, an autophagy inhibitor), respectively. As shown by the western blotting results, treatment with MG132 instead of BFA1 rescued the MIS12 protein abundance in FTO −/− hMPCs to a similar level with that in FTO +/+ hMPCs ( Fig. S2X and S2Y). Furthermore, treatment with MG132 restored the MIS12 protein levels that were further decreased upon cycloheximide (CHX) addition in FTO −/− hMPCs (Fig. 2J), indicating that FTO ablation accelerates MIS12 degradation via the proteasome pathway.
To further dissect the role of MIS12 in regulating hMPC senescence, we next ablated MIS12 in WT hMPCs using a lentivirus-mediated CRISPR/Cas9 knockout system (Figs. 2K and S2Z). We found that depletion of MIS12 accelerated hMPC senescence, as evidenced by compromised clonal expansion ability, reduced Ki67-positive cells, increased SA-β-gal activity, downregulated expression of Lamin B1, LAP2, and HP1α, and more abnormal nuclei, as well as shortened telomere length (Fig. 2L-S). Accordingly, these findings suggest that the presence of MIS12 counteracts acquisition of hMPC senescence.
In conclusion, we here used CRISPR/Cas9-based techniques to generate FTO-deficient hESCs and hMPCs, an approach through which we gained insights into the regulatory role of FTO and molecular mechanisms in homeostatic maintenance of human stem cells. As demonstrated by our data, FTO was dispensable for the maintenance of hESC pluripotency, consistent with a recent related study (Wei et al., 2021). In contrast, we demonstrated that ablation of FTO accelerated hMPC senescence, as manifested by decreased cell proliferation, impaired cell cycle and increased SA-β-gal activity. In line with our observations in FTO-deficient hMPCs, previous studies have unveiled that inhibition of FTO led to impeded cell cycle progression and compromised cell proliferation in myoblasts and a variety of cancer cells Deng et al., 2021). Moreover, FTO knockdown in human ovarian granulosa cells also resulted in accelerated senescence . Collectively, these reports and our study demonstrate celltype-specific roles of FTO. Cell cycle arrest is a common feature of cellular senescence (Wu et al., 2020). Consistently, we also detected aberrant cell cycle progress in FTO-deficient hMPCs. Furthermore, LC-MS/MS analysis identified a panel of cell cyclerelated FTO-interacting proteins, indicating a potential role of these cell cycle factors in regulating hMPC senescence upon FTO deficiency. Subsequently, we noticed MIS12, a cell cycle regulator that interacts with DSN1, NSL1, and PMF1, to form the kinetochore complex, thus promoting chromosome segregation during mitosis and being involved in the regulation of cell proliferation (Abe-Kanoh et al., 2019). Indeed, our previous study has demonstrated that downregulation of MIS12 due to m 6 A reduction decreased cell proliferation and accelerated senescence in hMPCs (Wu et al., 2020). Coincidentally, we also observed a decrease in the protein level of MIS12 in FTO-deficient hMPCs, and that knockout of MIS12 led to compromised self-renewal and premature senescence in WT hMPCs, mimicking majority of the senescent phenotypes caused by FTO deficiency. Discordantly, we did not detect any visible alterations in the amounts of both m 6 A modification and mRNA level of MIS12 upon FTO depletion, indicating an m 6 A-independent way by which FTO regulates the expression of MIS12. Apart from MIS12, NSL1, another component of the kinetochore complex (Abe-Kanoh et al., 2019), was also identified as a candidate protein interacting with FTO ( Fig. S2Q and S2R; Table S1), supporting the implication of cell cycle regulators in FTO-mediated biological process. Our further results demonstrated that FTO deficiency facilitated the degradation of MIS12 through the proteasome-mediated pathway. Although previous work supported that MIS12 can be degraded via the ubiquitin-proteasome system (Abe-Kanoh et al., 2019), how FTO protects MIS12 from degradation in hMPCs awaits further investigations. To be noted, though we demonstrated MIS12 as a downstream factor of FTO in regulating hMPC senescence, we could not rule out the possibility that there exist some other potential partners also involved in this process. For example, RB1 and CDKN2A, two well-known senescence regulators, were also identified as potential FTO-interacting proteins ( Fig. S2Q and Table S1). Whether RB1 or CDKN2A partially contributes to hMPC senescence caused by FTO deficiency needs to be explored.
Collectively, our study disclosed an uncharacterized role of FTO in maintaining the protein stability of MIS12, independent of its m 6 A demethylation activity, and in counteracting hMPC senescence. These new findings further increase the complexity of FTO in the regulation of cellular physiology. Yet, more efforts need to be integrated to figure out the possible explanation for the globally unchanged m 6 A abundance in FTO-deficient human stem cells.