Abstract
Craniofacial malformations, often associated with syndromes, are
prevalent birth defects. Emerging evidence underscores the importance
of m^6A modifications in various bioprocesses such as stem cell
differentiation, tissue development, and tumorigenesis. Here, in vivo,
experiments with zebrafish models revealed that mettl3-knockdown
embryos at 144 h postfertilization exhibited aberrant craniofacial
features, including altered mouth opening, jaw dimensions, ethmoid
plate, tooth formation and hypoactive behavior. Similarly, low METTL3
expression inhibited the proliferation and migration of BMSCs, HEPM
cells, and DPSCs. Loss of METTL3 led to reduced mRNA m^6A methylation
and PSEN1 expression, impacting craniofacial phenotypes. Co-injection
of mettl3 or psen1 mRNA rescued the level of Sox10 fusion protein,
promoted voluntary movement, and mitigated abnormal craniofacial
phenotypes induced by mettl3 knockdown in zebrafish. Mechanistically,
YTHDF1 enhanced the mRNA stability of m^6A-modified PSEN1, while
decreased METTL3-mediated m^6A methylation hindered β-catenin binding
to PSEN1, suppressing Wnt/β-catenin signaling. Pharmacological
activation of the Wnt/β-catenin pathway partially alleviated the
phenotypes of mettl3 morphant and reversed the decreases in cell
proliferation and migration induced by METTL3 silencing. This study
elucidates the pivotal role of METTL3 in craniofacial development via
the METTL3/YTHDF1/PSEN1/β-catenin signaling axis.
graphic file with name 41419_2024_6606_Figa_HTML.jpg
Subject terms: Developmental biology, Epigenetics
Introduction
Craniofacial malformations are prevalent birth defects that often
manifest as part of syndromes [[44]1, [45]2]. These defects encompass a
diverse array of phenotypes, such as cleft lip with or without cleft
palate, cleft palate alone, craniosynostosis, craniofacial microsomia,
and malocclusion [[46]3–[47]7]. The characterization of genetic and
epigenetic factors in patients with dysmorphologies and animal models
has illuminated the underlying etiological mechanisms contributing to
craniofacial developmental abnormalities.
N^6-methyladenosine (m^6A), the most abundant internal modification in
eukaryotic mRNAs, is critically important for various developmental
events [[48]8, [49]9]. Dynamic and reversible RNA methylation is
orchestrated by writer proteins (methyltransferases), eraser proteins
(demethylases) and reader proteins (RNA-binding proteins) [[50]10,
[51]11]. Methyltransferase-like 3 (METTL3), methyltransferase-like 14
(METTL14) and Wilms’ tumor 1-associated protein (WTAP) constitute the
core of methyltransferase complex [[52]12–[53]14]. The stable depletion
of m^6A through Mettl3 knockout in mice leads to embryonic lethality
and activates the pErk and pAkt signaling pathways, facilitating
pluripotency departure [[54]15, [55]16]. Mettl14 has been implicated in
the regulation of embryonic neural stem cell self-renewal and brain
development via histone modifications [[56]17]. Conversely, the fat
mass and obesity–associated protein (FTO) and alkB homolog 5 (ALKBH5)
serve as demethylases, reversing m^6A modifications. Recent studies
have revealed that FTO-mediated m^6A demethylation of LINE1 plays a
regulatory role in shaping the chromatin state and gene expression in
mouse oocytes and embryonic stem cells [[57]18].
Accumulating evidence underscores the crucial role of m^6A
modifications in shaping RNA fate and functions, including mRNA
stability, localization, splicing, transport, translation, microRNA
processing, and RNA-protein interactions. Moreover, m^6A modifications
are critical for diverse bioprocesses, ranging from stem cell
differentiation, tissue development, and sex determination to
tumorigenesis [[58]19–[59]24]. Despite this understanding, the
physiological role of m^6A modifications in abnormal craniofacial
development has not been fully elucidated.
In this study, we conducted in vivo and in vitro experiments using
zebrafish and cell models to explore the biological function of m^6A
methylase METTL3 involved in embryonic craniofacial development.
Furthermore, we observed a significant decrease in psen1 expression
upon mettl3 knockdown. Mechanistically, Mettl3-mediated m^6A
modification appeared to regulate the level of Sox10, possibly by
affecting psen1 in a YTHDF1-dependent manner, thereby influencing
Wnt/β-catenin signaling. Collectively, our findings provide compelling
evidence for the crucial involvement of the
METTL3/YTHDF1/PSEN1/β-catenin axis in vertebrate embryonic craniofacial
developmental events.
Results
METTL3 is involved in craniofacial development
We investigated the potential relevance of m^6A modifications to
developmental processes and congenital diseases. To identify critical
m^6A writers in this context, we conducted a comprehensive literature
search using PubMed, Google Scholar and Web of Science databases up to
August 2023. Each m^6A writer was assigned a score by summing the score
for the studies included (1 if it was reported, 0 if it was not
reported). Ultimately, 21 studies were identified, and among them,
METTL3 emerged with the highest score for m^6A modification, with a
score exceeding 10 (Supplementary Fig. [60]1A, Supplementary Table
[61]1). By analyzing spatial transcriptome data from C57BL/6 mouse
embryos at E16.5, we observed that the expression of Mettl3 was
predominantly clustered in regions associated with jaw, tooth, and
mucosal epithelium (Supplementary Fig. [62]1B). Furthermore, the
spatiotemporal mapping of developmental trajectories during zebrafish
embryogenesis at 12 h postfertilization (hpf) highlighted the
expression of mettl3 in neural crest clusters, a critical region that
has been linked to craniofacial development (Supplementary Fig.
[63]1C).
Expression pattern of Mettl3 in early zebrafish development
Bioinformatics analysis revealed that mettl3 encodes a 70 kDa protein
with a conserved catalytic DPPW motif (D399-W402), implying potential
methylation activity for zebrafish Mettl3 (Supplementary Fig. [64]2A).
Notably, the amino acid residue D395 in the S-adenosylmethionine (SAM)
binding motif is critical for methyltransferase (MTase) activity, and
W398 has been implicated in π–π stacking with the methylated adenine
base during substrate binding in Homo sapiens [[65]25]. Remarkably,
alignment analysis revealed that the residue D399 of Mettl3 in Danio
rerio exhibited evolutionary conservation with D395. This analysis also
highlighted a relatively high homology between zebrafish mettl3 and
human METTL3 (similarity, 68.3%).
Next, we investigated the expression pattern of mettl3 during various
zebrafish embryonic stages. Early expression of mettl3 in zebrafish
embryos decreased from 12 to 24 hpf (Supplementary Fig. [66]2B). These
data collectively suggest a potentially important role for Mettl3 in
zebrafish craniofacial development.
Mettl3 deficiency results in craniofacial abnormalities in zebrafish
To investigate the function of mettl3 in craniofacial development, we
generated a zebrafish model in which mettl3 was specifically knocked
down with morpholinos (MO). Injection of 8 ng of mettl3 MO led to
reduced mettl3 and m^6A levels, subsequently affecting embryo survival
and increasing the incidence of malformations (Fig. [67]1A–C).
Similarly, zebrafish embryos injected with mettl3 MO exhibited a
significant reduction in body length, heart-associated edema, and
craniofacial abnormalities when compared to the controls (Fig. [68]1D).
Notably, co-injection of mettl3 mRNA containing mettl3 MO target
sequence effectively rescued both mettl3 and m^6A levels, alongside
craniofacial phenotypes (Fig. [69]1A–D).
Fig. 1. Phenotypes of the zebrafish.
[70]Fig. 1
[71]Open in a new tab
A The expression of mettl3 in zebrafish embryos injected with control
morpholino (MO), mettl3 MO, or co-injection with mettl3 MO and mRNA at
48 hpf. B Representative dot blot showing m^6A levels in zebrafish
embryos injected with control MO, mettl3 MO, or co-injection with
mettl3 MO and mRNA. MB, methylene blue staining. C Statistical analysis
of the number of dead, abnormal or normal embryos. D Lateral view of
zebrafish larvae injected with control MO, mettl3 MO, or co-injection
with mettl3 MO and mRNA that were imaged with transmitted light at 48,
72, and 96 hpf. E–G Schematic diagram of zebrafish craniofacial
cartilage structures, including the distance of mouth opening, width
and length of the mandible, length of the palatoquadrate, and width and
length of the ethmoid plate, from lateral view and ventral views. H
Zebrafish embryos at 144 hpf were stained with alcian blue and alizarin
red to observe craniofacial structures. The red arrow shows the
development of tooth and pharyngeal in zebrafish embryos. I Scatter
histogram showing the length of the palatoquadrate, Meckel’s cartilage,
and the ethmoid plate; the width of Meckel’s cartilage and the ethmoid
plate; and the distance of mouth opening in zebrafish embryos injected
with control MO, mettl3 MO, or co-injection with mettl3 MO and mRNA
(each group, n = 100). J Iridophores at 48 and 72 hpf in zebrafish
embryos injected with control MO, mettl3 MO, or co-injection with
mettl3 MO and mRNA. Results were presented as mean ± SD of three
independent experiments. *P < 0.05, **P < 0.01 or ***P < 0.001
indicates a significant difference between the groups.
Building on these findings, we performed alcian blue and alizarin red
staining to explore craniofacial defects in mettl3 morphants. This
encompassed assessing parameters such as the mouth opening distance,
palatoquadrate and mandible length, as well as ethmoid plate length and
mandible and ethmoid plate width (Fig. [72]1E–G). Compared to control
morphants, the mettl3-knockdown embryos at 144 hpf exhibited increased
mouth opening distance and decreased palatoquadrate and mandible
lengths, analogous to the upper and the lower jaw structures,
respectively (Fig. [73]1H, I). Additionally, mettl3 morphants displayed
a shorter ethmoid plate length and width, analogous to the human palate
(Fig. [74]1H, I). Interestingly, significant changes in tooth formation
and a decrease in iridophores were detected in the mettl3 morphants
(Fig. [75]1H–J). Importantly, all these defects were also significantly
reversed by the expression of mettl3 mRNA (Fig. [76]1).
Furthermore, we explored the levels of Sox2 and Sox3, required for
early embryonic craniofacial differentiation in zebrafish. Notably, no
significant differences in Sox2 or Sox3 levels were observed in the
mettl3-knockdown zebrafish embryos at 72 or 96 hpf (Supplementary Fig.
[77]2C). Collectively, our data suggest that Mettl3 may exert a
critical role in embryonic craniofacial developmental processes,
possibly through its methylation activity in zebrafish.
Genetic associations in METTL3 related to craniofacial development-linked
diseases
Next, we evaluated the genetic associations of METTL3 with craniofacial
development-related diseases. In total, we identified four, six, and
six independent genetic variants in METTL3 associated with skeletal
sagittal malocclusion, hard palate cleft, and tooth development and
eruption, respectively (r^2 < 0.6, P < 5 × 10^−2) (Supplementary Table
[78]2). To provide insights into the underlying molecular mechanisms,
we highlight the variants with a relatively large effect. The rs1263800
and rs1263790 variants were associated with an increased risk for both
skeletal sagittal malocclusion and tooth development and eruption. We
embarked on a thorough extrapolation of the comprehensive catalog
detailing genetic influences on gene expression, wherein the rs1263800
variant demonstrated a significant association with METTL3 expression
in skeletal muscle (Supplementary Fig. [79]3A, B). Similarly, the risk
allele of rs1263790 displayed an association with reduced expression of
its corresponding gene METTL3 based on analysis of eQTLGen Consortium
data (z-score = −6.806, P = 1.00 × 10^−11).
Impact of low METTL3 expression on cell proliferation and migration
To further elucidate the functional roles of METTL3 in vitro, we
selected human bone marrow mesenchymal stem cells (BMSCs), human
embryonic palatal mesenchymal (HEPM) cells and human dental pulp
stromal cells (DPSCs), each of which corresponds to distinct
craniofacial phenotypes observed in zebrafish embryos or has been
linked to craniofacial development-linked diseases. A negative control
shRNA construct and two METTL3-specific shRNA constructs were designed.
Subsequently, qRT-PCR and western blot assays were performed to confirm
the successful knockdown of METTL3 in the cells (Fig. [80]2A, B). As
expected, METTL3 knockdown significantly reduced the m^6A levels (Fig.
[81]2C). Knockdown of METTL3 distinctly inhibited the proliferation
rate of the three cell types (Fig. [82]2D, E). Moreover, we found that
decreased METTL3 expression correlated with a notable decrease in cell
migration in transwell assays (Fig. [83]2F). Collectively, these data
suggested that low expression of METTL3 could effectively suppress the
proliferation and migration of BMSCs, HEPM cells and DPSCs,
highlighting the potential regulatory roles of METTL3 in craniofacial
development.
Fig. 2. METTL3 knockdown significantly suppresses cell proliferation and
migration in vitro.
[84]Fig. 2
[85]Open in a new tab
A, B The efficiency of METTL3 knockdown in BMSCs, HEPM cells, and
DPSCs. The expression of METTL3 was verified at both the mRNA and
protein levels. C Representative dot blot showing the m^6A levels in
cells with METTL3 knockdown and control groups. MB, methylene blue
staining. D, E Low METTL3 expression significantly reduced the
proliferation rate of BMSCs, HEPM cells, and DPSCs. F Low METTL3
expression significantly reduced the migration ability of BMSCs, HEPM
cells, and DPSCs. Results were presented as mean ± SD of three
independent experiments. *P < 0.05, **P < 0.01 or ***P < 0.001
indicates a significant difference between the designated groups.
Identification of METTL3 targets by high-throughput RNA-seq and m^6A-seq
To explore the mechanistic regulations of METTL3-induced m^6A
modification, we initially retrieved the MeRIP-seq and RNA-seq data
from mettl3 MO zebrafish embryos [[86]26] and performed RNA-seq
analysis of stable METTL3 knockdown or control BMSCs (Fig. [87]3A). The
MeRIP-seq analysis revealed hypomethylation of 6829 genes within
mettl3-deficient zebrafish embryos. Similarly, when comparing mettl3 MO
zebrafish embryos and controls, 6818 genes were found to be
differentially expressed (2356 upregulated and 4462 downregulated;
|log[2]FC | > 1.3, P < 0.05). Furthermore, METTL3 knockdown in BMSCs
resulted in the differential expression of 5098 genes (2714 upregulated
and 2384 downregulated; |log[2]FC | > 1.3, P < 0.05) (Fig. [88]3B).
This analysis ultimately revealed 76 overlapping downregulated genes
accompanied by hypomethylated m^6A peaks (Fig. [89]3C). Metascape
analysis showed that the 76 genes were primarily associated with somite
development, highlighting the crucial role of m^6A modification in
embryonic development (Fig. [90]3D). Kyoto Encyclopedia of Genes and
Genomes (KEGG) analysis revealed that the major affected pathways
encompassed Wnt signaling pathway and p53 signaling pathway (Fig.
[91]3E). Previous studies found that Wnt signaling is critical to early
embryo development and the biology of stem cells and progenitors
[[92]27, [93]28], thus we focused on the Wnt signaling pathway.
Fig. 3. Identification of METTL3 targets via MeRIP-seq and RNA-seq.
[94]Fig. 3
[95]Open in a new tab
A Schematic diagram depicting the protocols used for MeRIP-seq and
RNA-seq. B Volcano plot of differentially expressed genes between
METTL3 knockdown and control BMSCs. C Flow chart showing the shared
downregulated genes with hypomethylated m^6A peaks. D Gene enrichment
analysis performed via the Metascape database. E KEGG pathway
enrichment analysis shows major signaling pathways in METTL3-knockdown
BMSCs compared to control BMSCs. F The expression level of PSEN1 in
BMSCs, HEPM cells, and DPSCs in the METTL3-knockdown and control
groups. G The m^6A abundances of psen1 transcript in mettl3-knockdown
zebrafish embryos compared to control embryos. H Validation of m^6A
modification in METTL3-knockdown and control cells using MeRIP-qPCR.
Results were presented as mean ± SD of three independent experiments.
*P < 0.05, **P < 0.01 or ***P < 0.001 indicates a significant
difference between the groups.
To validate these findings, qRT‒PCR assays further confirmed the
reduction in the expression of only PSEN1, a component of the Wnt
signaling pathway, in METTL3-knockdown cells (Fig. [96]3F and
Supplementary Fig. [97]4A, B). Similarly, psen1 mRNA levels were
significantly lower in mettl3-knockdown zebrafish embryos than in
control zebrafish embryos (Supplementary Fig. [98]4C). scRNA-seq
analysis of expression data from bone marrow in the Tabula Sapiens
revealed that PSEN1 was expressed mainly in hematopoietic stem cells,
closely related to METTL3 expression (Supplementary Fig. [99]4D).
MeRIP-seq further unveiled a diminished m^6A peak in PSEN1 within
mettl3-deficient zebrafish embryos (Fig. [100]3G). Consistent with
these findings, MeRIP-qPCR confirmed that METTL3 silencing decreased
the m^6A levels in PSEN1 (Fig. [101]3H).
PSEN1 modulation by METTL3-mediated m^6A RNA methylation
Furthermore, we assessed the PSEN1 protein levels in METTL3 knockdown
cells, which exhibited significant downregulation (Fig. [102]4A). To
confirm our hypothesis, we employed the methylation inhibitor
3-deazaadenosine (DAA). Remarkably, treatment with DAA at varying
concentrations led to a considerable reduction in the PSEN1 levels
(Fig. [103]4B). To determine whether the regulatory effect of METTL3 on
PSEN1 is dependent on the methylation of its mRNA transcript targets,
we predicted potential m^6A sites in the full-length PSEN1 gene via
SRAMP (Fig. [104]4C, D) and constructed luciferase reporter plasmids
with the wild-type or mutant m^6A consensus sequence (GGACC to TTGAG)
in PSEN1 (Fig. [105]4E). Luciferase assays showed that only wild-type
PSEN1 significantly suppressed the transcription of PSEN1 in stable
METTL3 knockdown cells (Fig. [106]4F–H). To further verify the m^6A
modification sites at the PSEN1 transcript, the single-base elongation-
and ligation-based qPCR amplification method SELECT was used and the
probe pairs targeting the highly confident m^6A1585 site and A1579
control site at PSEN1 transcript respectively were designed. We
observed that m^6A1585 targeted site revealed significantly decreased
m^6A levels after METTL3 was knocked down (Fig. [107]4I–K).
Fig. 4. PSEN1 is modulated by METTL3-mediated m^6A RNA methylation.
[108]Fig. 4
[109]Open in a new tab
A The protein level of PSEN1 in BMSCs, HEPM cells and DPSCs with
METTL3-knockdown and control groups. B The relative expression of PSEN1
detected at the RNA level after treatment with DAA in at various
concentrations (0 μmol, 600 μmol and 700 μmol). C, D Potential m^6A
sites in full-length PSEN1 gene predicted using SRAMP. E The wild-type
or mutant m^6A consensus sequence fused with the firefly luciferase
reporter. F–H The transcription levels of wild-type and mutant PSEN1 in
BMSCs, HEPM cells and DPSCs. I–K The m^6A methylation level of the
PSEN1 at specific modification site (m^6A1585) and control site (A1579)
using SELECT in control and METTL3 knockdown cells. Results were
presented as mean ± SD of three independent experiments. *P < 0.05,
**P < 0.01 or ***P < 0.001 indicates a significant difference between
the groups.
Next, we determined whether METTL3 regulated cell proliferation and
migration via PSEN1 and found that PSEN1 overexpression partially
reversed the decreased proliferation and migration in METTL3-silenced
cells (Supplementary Fig. [110]5). We used bioinformatics to analyze
the spatial transcriptomics data of zebrafish embryogenesis at 18 hpf.
psen1 and mettl3 exhibited similar expression patterns, particularly in
the neural crest (Supplementary Fig. [111]6A). Additionally, early
psen1 expression was decreased in zebrafish embryos from 12 hpf to 24
hpf, which was closely correlated with mettl3 expression (Supplementary
Fig. [112]6B, C). To assess the involvement of psen1 in the regulation
of craniofacial abnormalities by mettl3 during zebrafish embryogenesis,
we designed a rescue experiment (Supplementary Fig. [113]6D).
Co-injection of psen1 mRNA significantly rescued abnormal craniofacial
phenotypes in mettl3 knockdown embryos (Fig. [114]5A–E). Moreover,
considering that neural crest cells (NCCs) play an important role in
zebrafish craniofacial development and that sox10 is expressed in
migrating zebrafish NCCs [[115]29], we used Tg(sox10:eGFP) transgenic
zebrafish embryos expressing green fluorescent protein (GFP) to study
the effects of mettl3 and psen1 during zebrafish embryogenesis.
Consistent with our prediction, the levels of both Sox10 fused to the
GFP protein and Sox10 were weak in the mettl3 morphants (Fig. [116]5F
and Supplementary Fig. [117]6E). Importantly, co-injection of mettl3 or
psen1 mRNA rescued the level of Sox10 fusion protein (Fig. [118]5F and
Supplementary Fig. [119]6F). Taken together, our results suggest that
Mettl3-mediated m^6A modification likely regulates the level of Sox10,
potentially via its effect on psen1, thereby influencing craniofacial
developmental events in vertebrate embryogenesis.
Fig. 5. psen1 rescues the level of Sox10 associated with migrating zebrafish
neural crest cells and abnormal craniofacial phenotypes.
[120]Fig. 5
[121]Open in a new tab
A, B Co-injection of psen1 mRNA rescued the abnormal craniofacial
phenotypes in mettl3-knockdown embryos. C Statistical analysis of the
number of dead, abnormal, or normal embryos. D Scatter histogram
showing the length of the palatoquadrate, Meckel’s cartilage, and the
ethmoid plate; the width of Meckel’s cartilage and the ethmoid plate;
and the distance of the mouth opening in zebrafish embryos injected
with control MO, mettl3 MO, or co-injection with mettl3 MO and psen1
mRNA (each group, n = 100). E Iridophores at 48, 72, and 96 hpf in
zebrafish embryos injected with control MO, mettl3 MO, or co-injection
with mettl3 MO and psen1 mRNA. F Tg(sox10: eGFP) transgenic zebrafish
embryos expressing green fluorescent protein (GFP) were used to explore
the effects of mettl3 and psen1 during zebrafish embryogenesis. Results
were presented as mean ± SD of three independent experiments.
*P < 0.05, **P < 0.01 or ***P < 0.001 indicates a significant
difference between the groups.
YTHDF1, an m^6A reader protein, maintains PSEN1 mRNA stability
Previous studies have indicated the important roles of m^6A reader
proteins in the regulation of RNA modifications [[122]10]. To identify
the m^6A reader that recognizes and binds PSEN1 for methylation, an RNA
pull-down assay employing biotin-labeled PSEN1 mRNA from BMSCs, HEPM
cells, and DPSCs was conducted (Fig. [123]6A). Mass spectrometry (MS)
analysis of the corresponding bands revealed that YTHDF1 emerged as the
prominent m^6A reader protein in three cell types (Supplementary Fig.
[124]7A–C, and Supplementary Table [125]3 to [126]5). This finding was
further confirmed by western blot analysis, which indicated direct
binding between YTHDF1 and PSEN1 mRNA in BMSCs, HEPM cells and DPSCs
(Fig. [127]6B).
Fig. 6. PSEN1 is specifically recognized by YTHDF1 and directly interacts
with β-catenin.
[128]Fig. 6
[129]Open in a new tab
A Schematic of the design for the RNA pull-down assay. B Immunoblotting
of YTHDF1 after the RNA pull-down assay with cell lysate,
biotinylated-PSEN1, and biotinylated-control in the cells. C Schematic
of the design for the RNA immunoprecipitation (RIP) assay. D RIP assay
to determine the enrichment of PSEN1 in cells incubated with
anti-YTHDF1 antibody. E–H Cells were transiently transfected with
control, siYTHDF1, empty vector, or OEYTHDF1, respectively. The
half-life (t[1/2]) of the PSEN1 mRNA was measured. I Silver staining
revealed PSEN1-bound proteins in BMSCs, HEPM cells and DPSCs. J
Interaction between β-catenin and PSEN1 determined by co-IP followed by
western blot analysis. K Representative image showing the enrichment of
PSEN1 and β-catenin after METTL3 knockdown by immunostaining analyses
in BMSCs, HEPM cells, and DPSCs. L Schematic diagram showing the
mechanism of PSEN1. Results were presented as mean ± SD of three
independent experiments. ***P < 0.001 indicates a significant
difference between the groups.
Next, the interaction between YTHDF1 and PSEN1 mRNA was assessed via
RNA immunoprecipitation (RIP) (Fig. [130]6C). The enrichment of PSEN1
PCR products was observed in YTHDF1 samples compared to the control
samples, validating the direct binding of YTHDF1 to PSEN1 mRNA (Fig.
[131]6D). As an important m^6A reader, YTHDF1 recognizes both G(m^6A)C
and A(m^6A)C RNAs as ligands without sequence selectivity and mediates
the expression of m^6A-modified target genes by enhancing the stability
of RNA [[132]30]. To measure the half-life of the PSEN1 transcript upon
the modulation of YTHDF1 expression, cells were treated with the
transcription inhibitor actinomycin D. Consistently, YTHDF1 knockdown
led to a significant decrease in the half-life of the PSEN1 transcript,
whereas YTHDF1 overexpression induced a noticeable increase in its
half-life (Fig. [133]6E–H). These findings collectively demonstrate
that METTL3-mediated m^6A modification controls PSEN1 expression by
regulating its mRNA stability in a YTHDF1-dependent manner.
PSEN1 interacts with β-catenin and regulates Wnt/β-catenin signaling via
METTL3-mediated m^6A modification
PSEN1 is a transmembrane protein that controls the activity of several
signaling pathways in subcellular compartments [[134]31]. To assess the
proteins that may interact with PSEN1, we performed
co-immunoprecipitation (co-IP) coupled with MS analysis and found that
five proteins could bind to PSEN1 in BMSCs, HEPM cells and DPSCs (Fig.
[135]6I, Supplementary Fig. [136]7D, and Supplementary Table [137]6 to
[138]11). Notably,β-catenin was the most likely protein to bind PSEN1
according to the protein-protein interaction (PPI) prediction with the
STRING and GEMANIA databases (Supplementary Fig. [139]7E, F).
Representative MS spectra of β-catenin are shown in Supplementary Fig.
[140]7G. Subsequent co-IP analysis, supported by western blot assays,
demonstrated the affinity isolated β-catenin for PSEN1 in cells (Fig.
[141]6J). To further validate the potential cooperation between
β-catenin and PSEN1 in the regulation of METTL3 expression, an
immunofluorescence (IF) assay was conducted, which revealed reduced
PSEN1 and β-catenin staining levels in stable METTL3 knockdown cells,
as compared to control cells. Additionally, colocalization of PSEN1 and
β-catenin was detected in the cytoplasm and nucleus of the above three
cell lines (Fig. [142]6K), which indicated that β-catenin could
directly bind to the PSEN1 protein (Fig. [143]6L).
Given that β-catenin is critical for the Wnt/β-catenin signaling
pathway, SuperTopFlash/SuperFopFlash reporters were constructed to
investigate whether METTL3 modulates this pathway by regulating PSEN1.
As expected, METTL3 knockdown markably decreased TOP/FOP
transcriptional activity, potentially inactivating Wnt/β-catenin
signaling (Fig. [144]7A–C). We next determined the protein levels of
several key genes within this pathway. The levels of β-catenin and TCF1
were decreased after METTL3 knockdown, while METTL3 knockdown
upregulated the level of GSK3 (Fig. [145]7D–F). Compared with that in
control cells, the phosphorylation of GSK3 in METTL3-knockdown cells
was significantly lower after normalization to total GSK3, but only a
decrease trend was detected for phosphorylation of β-catenin in METTL3
knockdown cells normalized to the total β-catenin (Fig. [146]7D–H).
Overall, reduced METTL3-mediated m^6A methylation can lead to a
decrease in the binding of the β-catenin to PSEN1, thus inhibiting
Wnt/β-catenin signaling.
Fig. 7. METTL3 deficiency inhibits Wnt/β-catenin signaling and Wnt/β-catenin
activation partially alleviates the phenotypes of mettl3 morphants.
[147]Fig. 7
[148]Open in a new tab
A–C Dual luciferase assay demonstrating the effect of SuperTop/SuperFop
reporter activity in BMSCs, HEPM cells, and DPSCs transfected with the
shMETTL3 vector. D–F Western blot showing the protein levels of TCF1,
GSK3, phosphorylated-GSK3, β-catenin, and phosphorylated-β-catenin in
control and stable METTL3 knockdown cells, GAPDH was used as a loading
control. G, H Quantitative analyses of the relative expression of
phosphorylated GSK3 and β-catenin. I Schematic of the experimental
design to assess the effect of CHIR99021 at different doses. J
Wnt/β-catenin activation partially alleviates the phenotypes of mettl3
morphants. Results were presented as mean ± SD of three independent
experiments. *P < 0.05, **P < 0.01 or ***P < 0.001 indicates a
significant difference between the groups.
Mitigation of disease-associated phenotypes through Wnt/β-catenin signaling
pathway activation by GSK3 treatment
We then explored whether pharmacological activation of Wnt/β-catenin
signaling could rescue the craniofacial malformations in the mettl3
morphants (Fig. [149]7I). Tg(sox10:eGFP) zebrafish larvae were treated
with CHIR99021, the most selective reported inhibitor of GSK3, at 15,
20 or 25 μM respectively [[150]32, [151]33]. The addition of CHIR99021
partially rescued NCC development and Sox10 expression in
mettl3-deficient embryos in a dose-dependent manner, whereas the
zebrafish embryos with mettl3 deficiency exhibited a weaker expression
of Sox10 accompanied by severe developmental abnormalities at
concentrations of 25 μM of CHIR99021 (Fig. [152]7J and Supplementary
Fig. [153]8A, B). Moreover, in vitro data showed that 2.5, 5 or 10 μM
CHIR99021 partially reversed the inhibition of cell proliferation and
migration induced by METTL3-silencing, especially at a concentration of
5 μM CHIR99021 (Supplementary Fig. [154]9-[155]10). These results
suggest that craniofacial anomalies in mettl3 morphants are associated
with downregulated Wnt/β-catenin signaling, which can be partially
alleviated by promoting Wnt/β-catenin signaling.
Mettl3 deficiency alters motor behavior in zebrafish larvae
As PSEN1 was reported to be critical for appropriate embryonic
neurogenesis [[156]34], we further confirmed whether the m^6A
modification of psen1 induced by Mettl3 affected the locomotor behavior
of zebrafish larvae by a photoperiod stimulation test (Supplementary
Fig. [157]11A). Records of larval motion trails showed that compared
with controls, mettl3 MO zebrafish embryos at 144 hpf exhibited
hypoactive behavior. In addition, mettl3 knockdown resulted in
decreases in total swimming distance and speed in comparison to those
in the control groups during dark-light transition stimulation
(Supplementary Fig. [158]11B–E). Remarkably, we found similar activity
maps after the co-injection of mettl3 or psen1 mRNA and treatment with
20 μM CHIR99021, which rescued the activity for voluntary movements.
Discussion
As the most common RNA modification, m^6A modification plays an
essential role in various diseases, spanning cancers to developmental
disorders [[159]35]. The dynamic regulation of m^6A methylation is
governed by three types of regulatory factors, methyltransferases,
demethylases, and reader proteins, which regulate RNA splicing,
translation, export, decay, and stability. Through bioinformatic
analyses and a literature search, METTL3 emerged as a potential
methyltransferase closely related to craniofacial development.
Operating within the methyltransferase complex in mammalian cells,
METTL3 methylates its specific target transcripts and participates in
various physiological processes encompassing embryonic development,
brain development, and cell reprogramming [[160]36, [161]37]. Cai et
al. reported that METTL3 directly interacts with ACLY and SLC25A1,
affecting the glycolytic pathway, and thus regulates the osteogenic
differentiation of DPSCs [[162]38]. Our present study explored the
outcomes of mettl3 knockdown, uncovering several craniofacial
developmental defects, including shorter palatoquadrate and mandible
lengths, decreased numbers of iridophores, aberrant tooth formation and
hypoactive behavior, in zebrafish embryos. Moreover, these phenotypes
were significantly rescued by mettl3 mRNA, underscoring the critical
role of METTL3 in embryonic craniofacial developmental processes.
Using MeRIP-seq and RNA-seq, the differentially expressed genes with
down-regulated m^6A levels were mainly related to somite development
and the Wnt signaling pathway via enrichment analysis. The formation of
somites during early embryogenesis is a fundamental and conserved
feature of all vertebrate species and results in the metameric
organization of the vertebrae and the associated skeletal muscles,
nerves, and blood vessels [[163]39]. Our in vivo experiments identified
PSEN1 within the Wnt signaling pathway as a pivotal target of METTL3.
Recent studies have characterized PSEN1 as a transmembrane protein with
nine transmembrane domains connected to hydrophilic loops in either the
extracellular space or the cytosol [[164]31, [165]40]. Cerebral
organoids derived from Alzheimer’s disease induced pluripotent stem
cell lines with mutant PSEN1 showed altered development and premature
differentiation during human stem cell neurogenesis [[166]41, [167]42].
However, the landscape of post-transcriptional PSEN1 mRNA regulation in
craniofacial development has not been determined. In this study, we
revealed the METTL3-PSEN1 mRNA interaction, underscoring the crucial
role of m^6A modification in regulating the PSEN1 level. Moreover,
PSEN1 overexpression partially reversed the inhibition of proliferation
and migration in METTL3-silenced cells. Co-injection of mettl3 or psen1
mRNA rescued mettl3 knockdown-induced hypoactive behavior in larvae.
These findings suggest a previously uncharacterized mechanism of PSEN1
in the regulation of m^6A modification.
Recent studies have indicated how m^6A modification affects transcript
translation, stability and intercellular transcriptome switching—a
pivotal mechanism in steering proper development [[168]22]. Neural
crest formation commences during early vertebrate development,
culminating in migration that contributes to a wide variety of
derivatives including craniofacial cartilage and bone, neurons of the
peripheral nervous system, and melanocytes [[169]43, [170]44]. As
master transcription factor of the neural crest, Sox10 is necessary for
the proliferation, migration, and differentiation of multipotent neural
crest during embryogenesis [[171]45, [172]46]. Consistent with the
expression of zebrafish sox10 in early cranial NCCs from 10 to 16 hpf
[[173]47], both mettl3 and psen1 exhibited similar expression patterns
based on the spatial transcriptomics data obtained during zebrafish
embryogenesis and early gene expression detection. Notably, the rescue
of Sox10 expression alongside abnormal craniofacial phenotypes induced
by mettl3 or psen1 mRNA indicates that mettl3-mediated m^6A
modification regulates Sox10, possibly through its effect on psen1,
thereby influencing craniofacial developmental events in vertebrate
embryogenesis.
The well-established role of m^6A modification in the regulation of
mRNA decay, translation efficiency, mRNA splicing, and export by
binding to the reader protein has been extensively described [[174]48].
RNA pull-down and RIP assays demonstrated that YTHDF1, but not the
other readers, could bind to PSEN1 mRNA. YTHDF1 is an important m^6A
modification reader, and its primary function is to promote protein
translation or regulate the stability of m^6A-modified mRNAs [[175]49,
[176]50]. Notably, YTHDF1 knockdown attenuated ameliorated pulmonary
artery smooth muscle cell proliferation, phenotypic switching, and the
development of pulmonary hypertension by enhancing MAGED1 translation
both in vivo and in vitro [[177]51]. Another study found that YTHDF1
enhanced the stability of c-Myc mRNA catalyzed in an m^6A-dependent
manner, thereby promoting c-Myc expression [[178]24]. Our results are
consistent with this mechanism, as we found that the m^6A modification
of PSEN1 enhanced mRNA stability in a YTHDF1-dependent manner. The
current study substantiates the ability of METTL3 to regulate the m^6A
modification of PSEN1, while YTHDF1 enhances the mRNA stability of
m^6A-modified PSEN1.
The crucial role of β-catenin within Wnt1 expressing cell lineages has
been well established, as its inactivation has been correlated with
severe craniofacial defects [[179]52, [180]53]. The potential
involvement of PSEN1 in Wnt signaling through the regulation of
β-catenin stability positions it as a candidate for in-depth
mechanistic exploration [[181]40]. In light of its central role,
β-catenin serves as an intracellular messenger for canonical Wnt
signaling, a pathway instrumental in activating and maintaining the
factors necessary for NCC development [[182]54, [183]55]. Similarly,
the significant reduction of β-catenin in stable METTL3 knockdown cells
impeded Wnt pathway activation, concurrently suppressing Sox10
expression via METTL3-YTHDF1-dependent silencing of PSEN1 mRNA.
Importantly, pharmacological activation of the Wnt/β-catenin pathway
partially alleviated the phenotypes of mettl3 morphants, rescued the
activity of voluntary movements and reversed the decreases in cell
proliferation and migration induced by METTL3 silencing, thereby
reinforcing the role of Wnt signaling in this process.
In summary, we identified and characterized PSEN1 as a novel
m^6A-regulated target in craniofacial development. Mechanistically,
METTL3-mediated m^6A modification controls the expression of PSEN1 by
regulating its mRNA stability in a YTHDF1-dependent manner. PSEN1
regulates Wnt/β-catenin signaling by binding to β-catenin, thereby
influencing craniofacial developmental events via METTL3-mediated m^6A
modification. Our study highlights the functional importance of the
METTL3/YTHDF1/PSEN1/β-catenin signaling axis, which provides new
insights into the exploration of the underlying regulation mechanism
for craniofacial development.
Materials and methods
Zebrafish maintenance
Zebrafish (Tubingen) and Tg(sox10:eGFP) transgenic zebrafish were
maintained at 28 °C using standard protocols. Zebrafish embryos were
cultured in E3 medium supplemented with 0.01 mg/L methylene blue and
collected at the 1-cell stage. All the experimental protocols were
approved by the Animal Ethics Committee of the Affiliated Stomatology
Hospital of Nanjing Medical University. For all zebrafish experiments,
embryos were collected and sorted into treatments blindly to ensure
appropriate randomization.
MO-mediated knockdown, rescue, and treatment with the GSK3 inhibitor
CHIR99021
MO antisense oligonucleotides targeting mettl3 (forward:
5’-ACCCAGAGCTAGAGAAGAGG-3’, reverse: 5’-CACAGAACTCCTGAACTTGA-3’, RC:
TCAAGTTCAGGAGTTCTGTG) were synthesized by GeneTools to block the
translation of mettl3 mRNA. A 5-base-pair mismatch MO served as a
control. The mettl3 MO (8 ng) or control MO (8 ng) was microinjected
into zebrafish embryos at 1-cell stage as previously described
[[184]56]. For rescue experiments, zebrafish mettl3 or psen1 mRNA
vectors (100 ng/μl) were co-injected with mettl3 MO at 1-cell stage.
Knockdown and rescue of mettl3 were further confirmed by qRT-PCR.
Tg(sox10:eGFP) zebrafish larvae injected with the mettl3 MO were
exposed to GSK3 inhibitor CHIR99021 (15, 20 or 25 μM) during zebrafish
embryonic development [[185]32, [186]33]. Embryos were imaged using a
Nikon SMZ800N stereomicroscope (NIKON Corporation, Tokyo, Japan).
Alcian blue and alizarin red staining
To assess morphological craniofacial changes, zebrafish embryos at
144 h post-fertilization (hpf) were randomly collected and fixed in 95%
ethanol overnight. Subsequent staining involved incubating the embryos
with 0.02% alcian blue (A8140, Solarbio, Beijing, China) and 0.5%
alizarin red overnight, followed by soaking in distilled water for
10 min and bleaching with 1.5% H[2]O[2]/1.5% KOH for 3 h until soft
tissue transparency was achieved. The embryos were washed twice in 50%
glycerol at room temperature. Phenotypic assessments included
quantitative analysis of mouth opening distance, palatoquadrate and
mandible lengths, as well as ethmoid plate and mandible cartilage
widths.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from zebrafish embryos and cells using an RNA
Extraction Kit following the manufacturer’s instructions (RX112,
Vazyme, Nanjing, China). cDNA templates were synthesized from RNA using
the PrimeScript™ RT Regent Kit (RR036A, Takara Bio, Shiga, Japan).
qRT-PCR analysis was performed with a SYBR Green RT-PCR Kit (Q712-02,
Vazyme, Nanjing, China) and a Roche Light Cycler 480 II system (Roche,
Switzerland). The sequences of primers used are listed in Supplementary
Table [187]12. GAPDH or gapdh was used as the internal control in all
the experiments.
m^6A dot blot assay
Total RNA from zebrafish embryos or cells was denatured at 95 °C for
5 min and spotted onto nylon membranes (GE Healthcare, USA). After
ultraviolet crosslinking, the membranes were incubated with an m^6A
antibody (1:250, CST, #56593) overnight. Following incubation with
horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody
for 1 h at room temperature and enhanced chemiluminescence development,
the membranes were dyed with 0.02% methylene blue (M196499, Aladdin,
Shanghai, China) and 1× TAE (ST716, Beyotime, China) and scanned to
determine the input RNA content.
Western blot assay
Total protein was extracted from zebrafish embryos and cultured cells
with RIPA lysis buffer supplemented with 0.5% PMSF and protease
inhibitors. Equal amounts of protein were separated on 10% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) and
transferred to polyvinylidene fluoride (PVDF) membranes (Millipore,
Darmstadt, Germany). The membranes were blocked with 5% non-fat milk
for 2 h at room temperature and then immunostained with primary
antibodies overnight at 4 °C. The primary antibodies used in this study
were as follows: anti-Sox2 (1:1000, GeneTeX, #GTX627404), anti-Sox3
(1:1000, GeneTeX, #GTX132494), anti-Sox10 (1:1000, GeneTeX,
#GTX128374), anti-β-actin(1:1000, GeneTeX, #GTX629630), anti-METTL3
(1:1000, Abcam, ab195352), anti-PSEN1 (1:1000, CST, #5643), anti-YTHDF1
(1:1000, Proteintech, 17479-1-AP) and anti-GAPDH (1:1000, Beyotime,
AG019), anti-TCF1 (1:1000, CST, #2203 T), anti-phospho-β-catenin
(1:1000, CST, #9561), anti-GSK3(1:5000, Abcam, ab40870), and anti-GSK3
phospho Y216 + Y279 (1:1000, Abcam, ab68476). Subsequently, the
membranes were incubated with the corresponding secondary antibodies
for 1 h, and washed with TBST three times, followed by detection with
the ECL chemiluminescent detection system ([188]P10100, NcmECL Ultra,
Suzhou, China).
Data sources
Processed data from spatiotemporal transcriptomic atlases of mouse
organogenesis were obtained from MOSTA
([189]https://db.cngb.org/stomics/mosta) [[190]57]. Dynamic
spatiotemporal transcriptomic atlas during zebrafish embryogenesis was
downloaded from the ZESTA database
([191]https://db.cngb.org/stomics/zesta/) [[192]58]. The single-cell
dataset from bone marrow in the Tabula Sapiens database was available
from the Gene Expression Omnibus (accession numbers [193]GSE201333).
Study cohort, genotyping, quality control, and phenotyping
The study cohort comprised a skeletal sagittal malocclusion cohort that
underwent genotyping using the Illumina Global Screen Array (GSA) by
Genergy Biotechnology Co. Ltd, that included 471 cases (0.7
[MATH: °≤ :MATH]
ANB
[MATH: ≤ :MATH]
4.7°) and 664 healthy controls (ANB < 0.7° or ANB > 4.7°) [[194]59,
[195]60]. Additionally, FinnGen summary statistics, encompassing tooth
development and eruption data, and information on hard palate cleft,
were imported from a researcher-accessible source
([196]https://r9.finngen.fi) in July 2023. Variants and samples were
excluded under the following conditions: (1) call rates less than 95%.
(2) minor allele frequency (MAF) lower than 0.01 and (3) genotype
distribution deviating from Hardy-Weinberg equilibrium
(P < 1.00 × 10^−5). All the study protocols were approved by the
Affiliated Stomatology Hospital of Nanjing Medical University
(PJ2020-079-001).
Cell culture, lentiviral transduction, expression plasmids, short interfering
RNAs, cell transfection and treatment
Human bone marrow mesenchymal stem cells (BMSCs) were purchased from
Cyagen Biosciences (Guangzhou, China). Adherent BMSCs were cultured in
BMSC growth medium (Cyagen Biosciences, Inc., Guangzhou, China) at
37 °C with 5% CO[2] and were passaged after reaching 80% confluence.
Human embryonic palatal mesenchymal (HEPM) cells were cultured in alpha
Dulbecco’s modified Eagle’s medium (α-MEM) containing 10% fetal bovine
serum (Gibco, USA), 100 μg/ml streptomycin (Gibco, USA), and 100 U/ml
penicillin (Gibco, USA) at 37 °C in 5% CO[2]. Similarly, human dental
pulp stromal cells (DPSCs) were cultured in α-MEM containing 10% fetal
bovine serum, 100 μg/ml streptomycin, and 100 U/ml penicillin at 37 °C
in 5% CO[2]. Cells from passages 2-7 were used in subsequent
experiments.
For stable knockdown, shRNA oligos targeting METTL3 were designed
(GenePharma, Shanghai, China). The lentivirus containing LV3-GFP was
purchased and packaged according to the manufacturer’s instructions.
The indicated cell lines were transduced with the shMETTL3-1/shMETTL3-2
or empty vector lentiviruses containing medium for 48 h, followed by
puromycin selection.
Small interfering RNA (siRNA) duplexes targeting human YTHDF1 were
synthesized by GenePharma (China). Human PSEN1 ([197]NM_000021.4) and
YTHDF1 ([198]NM_017798.4) was cloned through PCR and constructed in the
pEX-3 vectors for overexpression respectively. The designated siRNAs
and plasmids were transfected into cells with Lipofectamine 2000
(Invitrogen, USA) according to the manufacturer’s protocols.
Thereafter, the transfection efficacy was tested by qRT-PCR and western
blotting. The sequences of the siRNAs, vectors, and shRNAs used are
listed in Supplementary Table [199]12.
To explore the impact of pharmacological activation of Wnt/β-catenin
signaling on cell phenotypes, the cells were treated with the GSK3
inhibitor CHIR99021 (2.5, 5 or 10 μM) based on previous studies
[[200]61, [201]62].
Cell proliferation and migration assays
To ascertain cell proliferation, approximately 3000 transfected cells
were seeded in 96-well plates as monolayers. Cell viability was
measured at various time points using the Cell Counting Kit-8 (CCK8,
Kumamoto, Japan) on a microplate reader (SpectraMax 190, Molecular
Devices, USA) at 450 nm (OD450). Moreover, EdU reagent was used for
proliferation assessment following the manufacturer’s instructions.
Fluorescence microscope (Leica, DM4000) was utilized for sample
analysis. Three replicates were performed for each sample.
Additionally, transwell assays were performed to evaluate cell
migration. The cell suspension was seeded in the upper layer of the
transwell chambers (8 μm pore size, Millipore, Darmstadt, Germany) in
serum-free α-MEM. The lower chambers contained 600 μl of complete α-MEM
containing 20% FBS. After 24-36 h of incubation, cells that migrated
through the membrane were fixed with 4% paraformaldehyde, stained with
crystal violet, and counted in four randomly chosen fields under a
microscope (Nikon, SMZ800N, Tokyo, Japan).
Public sequencing datasets and RNA-seq
The MeRIP-seq and RNA-seq datasets of mettl3 morphants, accessible
through the Gene Expression Omnibus (GEO) public database under
accession number [202]GSE89655, were analyzed using methodologies
consistent with those used in a previous study [[203]26]. To ensure
data quality, we employed FastQC and RseQC to verify the sequence
quality. Subsequently, the HISAT2 tool was utilized for the alignment
of reads to the reference genome from Danio rerio (GRCz10). Peak
calling and differential peak analysis were performed with the R
package exomePeak2, and peak annotation was performed through
intersection with gene architecture using the R package ANNOVAR. For
the RNA-seq analysis of mettl3 morphants, after undergoing quality
control, the clean reads were mapped to each Ensembl gene (GRCz10). Raw
reads were generated via illumina Hiseq. Differential gene expression
was assessed using the DESeq2 package in R software.
For the RNA-seq assay, total RNA was extracted from control or
METTL3-knockdown BMSCs and then subjected to library construction and
sequencing. After quality control, clean reads were mapped to human
reference genome (hg38) and raw counts were generated via Illumina
NovaSeq. Differentially expressed genes (DEGs) were selected based on
the following criteria: |log[2](fold change) | >1.3 and P value < 0.05.
The volcano map of DEGs was generated with the R package ggplot2
v3.4.2.
Functional enrichment analysis
Enrichment analysis of DEGs with downregulated m^6A levels was analyzed
based on Metascape online tool
([204]http://metascape.org/gp/index.html#/main/step1). For the
identification of perturbed pathways, KEGG pathway analysis was
performed by employing the clusterProfiler package, with a significance
threshold of P < 0.05 based on marker genes.
MeRIP-qPCR
To quantify m^6A-modified specific gene levels, total RNA was extracted
from cells. A portion of the RNA sample was reserved as the input
control. The remaining RNA was subjected to an overnight incubation at
4 °C with anti-m^6A-antibody-conjugated or IgG-conjugated beads in
immunoprecipitation buffer supplemented with RNase inhibitor. The
m^6A-containing RNA samples were immunoprecipitated and eluted from the
beads. Subsequently, both the input and IgG control samples, alongside
the m^6A-immunoprecipitated samples were isolated and purified for qPCR
analysis, facilitating the detection of the target mRNAs.
DAA treatment
To evaluate the functional impact of METTL3-mediated m^6A modification
on the regulation of PSEN1, we employed the methylation inhibitor DAA
[[205]63]. Cells were cultured in 6-well plates and treated with
various concentrations of DAA (600 and 700 μmol). Cells were harvested
48 h after DAA treatment and total RNA was extracted for qRT-PCR
assays.
Motif analysis
We employed SRAMP ([206]http://www.cuilab.cn/sramp), a powerful tool
for predicting m^6A modifications at different loci with varying
confidence thresholds. This allowed us to predict the motif of m^6A
modifications [[207]64].
Luciferase reporter assay
To evaluate the impact of m^6A sites on PSEN1 expression, cDNA
containing the 3’UTR sequence of PSEN1 was inserted into a luciferase
reporter vector (pcDNA3.1 vector) as the wild-type construct. Predicted
m^6A sites (sequence: GGACC) in PSEN1 were replaced with random
sequence (TTGAG) to generate the mutant plasmids. METTL3-knockdown and
control cells were transfected with wild-type or mutated PSEN1 reporter
plasmids. After 48 h of transfection, we measured the relative
luciferase activity using the Duo-Lite Luciferase Assay System
(DD1205-01, Vazyme, Nanjing, China). All the experiments were performed
in triplicate.
SELECT assay for the single-base detection of m^6A modification
To detect the m^6A modification at the single-site level, we applied
the single-base elongation- and ligation-based qPCR amplification
method (SELECT) [[208]65]. The SELECT assays were performed with an
Epi-SELECT™ m^6A Site Identification Kit (Epibiotek, Guangzhou, China)
[[209]66]. In total, the probe pairs targeting the sites at the PSEN1
transcript and primers used for the SELECT assays are listed in
Supplementary Table [210]12. The qPCR reaction was performed using
ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711) with the Roche
Lightcycler 480 Instrument II system.
RNA pull-down and mass spectrometry (MS) analysis
Specific probes for PSEN1 and corresponding antisense probes were
synthesized by GenePharma (China). Total protein was extracted from the
cells. The RNA pull-down assay was performed according to the
manufacturer’s instructions using the Pierce Magnetic RNA–Protein
Pull-Down Kit (Thermo Fisher Scientific). Co-precipitated proteins were
eluted from the beads using loading buffer. The recruited proteins were
visualized by silver staining using the Fast Silver Stain Kit and
further characterized through MS analysis. The expression of the
identified proteins was confirmed by western blotting.
RIP assays
We conducted RIP assays using the Magna RIP kit (17-700, Millipore
Magna, USA) according to the manufacturer’s guidelines. Approximately
1 × 10^7 cells were collected and lysed with lysis buffer. The
supernatant was incubated with magnetic beads coupled with either
anti-YTHDF1 antibody (Proteintech, 17479-1-AP) or control IgG at 4 °C
overnight. The bead-RNA-protein complex was washed and treated with
Proteinase K. The precipitated RNA was further purified and quantified
by qRT-PCR.
RNA stability assay
YTHDF1-knockdown, YTHDF1-overexpression, and control cells were exposed
to 5 μg/mL actinomycin D (M4881, Abmole Bioscience, USA) to suppress
mRNA transcription. Cells were collected at various time points (0, 2,
4, 6, 8, and 10 h posttermination) and total RNA was extracted. The
remaining PSEN1 mRNA level was quantified using qRT-PCR. Half‐life
(t[1/2]) of PSEN1 mRNA was calculated according to previous methods
[[211]17, [212]67] and GAPDH was used for normalization.
Co-IP
Cells were harvested and resuspended with ice-cold IP lysis buffer
(Thermo Fisher Scientific, 88804). The lysates were centrifuged at
14,000 rpm for 10 min to obtain the supernatant containing total
proteins. Then the cell lysate was mixed with PSEN1 or CTNNB1 antibody.
After gently rotating at 4 °C overnight, 5 μl protein A/G agarose beads
were added to each tube and incubated at 4 °C for 1–3 h. Finally, the
beads were rinsed with wash buffer and heated at 95 °C for 10 min.
After silver staining using the Fast Silver Stain Kit, MS analysis was
carried out to identify substrate proteins that bind to PSEN1 based on
their scores and masses. Western blot assay was performed to validate
the interactions between bound proteins and PSEN1.
IF staining
The METTL3-knockdown and control cells were seeded onto cover slides
and incubated under standard culture conditions for 24–36 h. After an
overnight culture, cells were fixed in 4% paraformaldehyde for 15 min
at room temperature and then permeabilized with 0.1% Triton X-100.
Subsequently, cells were incubated with appropriate primary antibodies
(anti-PSEN1 1:50, CST, #5643 and anti-β-catenin 1:50, Santa Cruz,
sc-7963) at 4 °C overnight and subjected to secondary antibodies
conjugated with Alexa Fluor (Invitrogen) for 1 h at room temperature.
DAPI containing antifade medium was used for nuclear staining. Images
were captured by Nikon Imaging Software (NIS)-Elements and evaluated
using ImageJ software.
SuperTopFlash reporter assay
The SuperTopFlash/SuperFopFlash reporter assay was used to measure
β-catenin–driven TCF/LEF transcriptional activation. The cells were
co-transfected with the SuperTopFlash/SuperFopFlash construct
(Beyotime, China), and either the METTL3 knockdown vector or the
control vector using Lipofectamine 2000 transfection reagent (Life
Technologies). After 48 h post-transfection, cells were lysed and
luciferase activity was evaluated using a dual luciferase reporter
system. Firefly luciferase activity normalized to Renilla luciferase
activity was expressed as the relative fold change.
Behavioral assays
After incubating for 144 h, the zebrafish larvae in each treatment
group were transferred to the 24-well plates and the behaviors
including trajectories, swimming distance, and swimming speed were
assessed via a DanioVision observation chamber (Noldus Information
Technology, Netherlands). Zebrafish larvae were acclimated to plate
conditions for 30 min before the behavioral analysis. The 20 min
light-induced visual motor response recording consisted of two
alternate cycles of 5 min in the dark followed by 5 min in light. The
temperature of the well plates (28 ± 0.5 °C) during the experiment was
maintained by the DanioVision temperature control unit. All the video
recordings were analyzed using the EthoVision XT software (Noldus
Information Technology, Netherlands).
Statistical analysis
All numerical data are presented as means ± standard deviations (SD).
Pilot experiments and previously published results were used to
estimate the proper sample size. Experiments were independently
repeated at least three times. Differences between two groups or
multiple groups were evaluated by Student’s t‐test, Mann-Whitney U-test
and ANOVA test. Pearson’s correlation analysis was utilized to describe
correlations between quantitative variables without a normal
distribution. The ORs and 95% CIs for genetic associations were
obtained using multivariate logistic regression analysis. Statistical
analyses were calculated with R software (version 4.0.5) and the
figures were plotted using GraphPad Prism 7.0 (GraphPad Software, La
Jolla, CA, USA). All statistical tests were two‐sided. A two‐sided
P-value of less than 0.05 was considered to indicate statistical
significance (*P-value < 0.05, **P-value < 0.01, ***P-value < 0.001;
ns, not significant).
Supplementary information
[213]Supplementary Figures^ (51.6MB, pdf)
[214]Supplementary Table 1^ (10KB, xlsx)
[215]Supplementary Table 2^ (11.1KB, xlsx)
[216]Supplementary Table 3^ (16.5KB, xlsx)
[217]Supplementary Table 4^ (15.3KB, xlsx)
[218]Supplementary Table 5^ (27.7KB, xlsx)
[219]Supplementary Table 6^ (83.5KB, xlsx)
[220]Supplementary Table 7^ (73.2KB, xlsx)
[221]Supplementary Table 8^ (85.7KB, xlsx)
[222]Supplementary Table 9^ (71.6KB, xlsx)
[223]Supplementary Table 10^ (35KB, xlsx)
[224]Supplementary Table 11^ (16.1KB, xlsx)
[225]Supplementary Table 12^ (10.8KB, xlsx)
[226]checklist^ (1.8MB, pdf)
Author contributions
LM and YP conceptualized and supervised this study. LM, XZ, and SY
performed most experiments. YZ, JM, XZ, and LF contributed to data
collection and bioinformatics analyses. BV, SL, DL, and LW provided
technique support. LM, XZ, SY, and YP prepared the manuscript. All
authors reviewed the manuscript.
Funding
This work was supported by the National Natural Science Foundation of
China (81830031, 81970969, 82001088, 82101054, 82270496), the Natural
Science Foundation of Jiangsu Province (BK20220309), the Natural
Science Foundation of the Jiangsu Higher Education Institutions of
China (22KJB320003, 22KJA320002), Chinese Postdoctoral Science
Foundation (2022M721677), Young Talents Project of the Orthodontics
Committee of the Chinese Stomatological Association (COS-B2021-09),
Jiangsu Province Capability Improvement Project through Science,
Technology and Education-Jiangsu Provincial Research Hospital
Cultivation Unit (YJXYYJSDW4), Jiangsu Provincial Medical Innovation
Center (CXZX202227), as well as the German Research Foundation DFG VO
2138/7-1 grant 469177153 (BV), through the Collaborative Research
Center 889 and the Multiscale Bioimaging Cluster of Excellence (MBExC).
Data availability
All data needed to evaluate the conclusions in the paper are present in
the paper and/or the Supplementary Materials. Accession numbers of
published data used in this study are [227]GSE201333 and [228]GSE89655.
Competing interests
The authors declare no competing interests.
Ethics approval
This study was approved by the Nanjing Medical University Institution
Review Board. All animal experiments were approved by the Animal Ethics
Committee of the Affiliated Stomatology Hospital of Nanjing Medical
University.
Footnotes
Edited by Professor Mauro Piacentini
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
These authors contributed equally: Lan Ma, Xi Zhou, Siyue Yao.
Supplementary information
The online version contains supplementary material available at
10.1038/s41419-024-06606-9.
References