Graphical abstract

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Highlights

     * •
       Arhgef2 mediates total m^6A level via Mettl14
     * •
       Arhgef2 affects m^6A methylations of the Npdc1 and Cend1 mRNAs
     * •
       Decreased m^6A methylations inhibits translation of Npdc1 and
       nuclear export of Cend1
     * •
       Reduced protein expression of Npdc1 and Cend1 hinders neural
       differentiation
     __________________________________________________________________

   Molecular neuroscience; Developmental neuroscience; Cellular
   neuroscience

Introduction

   ARHGEF2 (synonym GEF-H1, murine Lfc), a member of the Rho guanine
   nucleotide exchange factor family, is crucial for controlling the
   spatiotemporal activation of Rho GTPases ([64]Ren et al., 1998;
   [65]Krendel et al., 2002). It is now well established that ARHGEF2
   controls microtubule dynamics through RhoA activation ([66]Meiri
   et al., 2012; [67]Birkenfeld et al., 2007; [68]Tonami et al., 2011).
   Lack of ARHGEF2 is strongly associated with an alternation in the
   organization of the actin cytoskeleton at the cell edge ([69]Nalbant
   et al., 2009). ARHGEF2 is required for mitotic spindle formation and
   orientation ([70]Bakal et al., 2005; [71]Yoshizaki et al., 2004).
   Recently, we successfully linked ARHGEF2 to neurologic disease by
   reporting a homozygous ARHGEF2 mutation in individuals with a
   neurodevelopmental phenotype including brain malformation
   ([72]Ravindran et al., 2017). ARHGEF2 loss of function impaired
   neuronal migration by RhoA/ROCK/MLC pathway, which caused the abnormal
   location of precerebellar neurons in the hindbrain ([73]Ravindran
   et al., 2017). Although we found that defective ARHGEF2 resulted in
   reduced neurogenesis of neocortical progenitors and probably
   contributed to microcephaly phenotype, little is known about whether
   ARHGEF2 regulates neurite outgrowth and synaptic formation during
   neural differentiation. Thus, the molecular mechanism that how ARHGEF2
   mediates neural differentiation of brain cortex needs to be further
   investigated.

   Dynamic methylation at the N^6 site of adenosine (N^6-methyladenosine,
   m^6A) has been indicated as the most prevalent internal mRNA
   modification in eukaryotes ([74]Zhuang et al., 2019). A growing body of
   evidence has revealed that 0.1%–0.4% of all adenosines in mammal
   transcripts are modified by m^6A, which enriched in long exons, near
   transcription start sites, and stop codons ([75]Meyer and Jaffrey,
   2014; [76]Wang and Zhao, 2016). It is involved in various biological
   processes, including mRNA splicing, localization, stability, and
   translation ([77]Haussmann et al., 2016; [78]Roundtree et al., 2017;
   [79]Wang et al., 2014; [80]Li et al., 2017). In recent years, m^6A is
   emerging as a key factor regulating neural differentiation. Deficiency
   of methyltransferase-like Mettl14 leads to the reduced nuclear export
   of m^6A-modified mRNAs regulating neural differentiation ([81]Edens
   et al., 2019). Depletion of m^6A by Mettl14 knockout in embryonic mouse
   brains prolongs the cell cycle of radial glia cells and extends
   cortical neurogenesis into postnatal stages ([82]Yoon et al., 2017),
   phenocopying Arhgef2 knockout mice we constructed in this study.
   Furthermore, in view of drastically increased m^6A levels in adult
   mouse brain compared with embryogenesis period ([83]Meyer et al.,
   2012), it remains possible that m^6A modification plays a unique role
   in the adult brain. Indeed, a compelling evidence points out that
   deletion of Mettl14 in the adult brain severely impairs learning and
   performance, which is correlated to protein synthesis in synapses of
   striatum ([84]Koranda et al., 2018). However, the mechanism of m^6A
   modification on neurite outgrowth and synaptic formation in postnatal
   brain cortex is still covered with a mysterious veil.

   In this study, we cross-linked the function of Arhgef2 and modification
   of m^6A regulated by Mettl14. The anomaly of neurogenesis, neurite
   outgrowth, and synaptic formation was demonstrated in an Arhgef2
   knockout mouse model. We further illustrated the function of Mettl14 in
   this process and investigated underlying cellular and molecular
   mechanisms. Together, our data revealed a vital role of Arhgef2 in
   regulating neural differentiation by m^6A-modified target mRNAs.

Results

Defective Arhgef2 caused microcephaly in adult mice

   Previously, we reported a homozygous ARHGEF2 mutation in individuals
   with a neurodevelopmental phenotype including brain malformation
   ([85]Ravindran et al., 2017). To explore the pathomechanism of brain
   malformation by loss of Arhgef2, we constructed an Arhgef2 knockout
   (Arhgef2−/−) mice model using CRISPR-Cas9 genome editing technology
   ([86]Figure S1). A 1.14-fold decrease in brain weight was noted in
   response to Arhgef2 knockout, whereas the body weight had no
   significant change. Compared with wild-type littermates, the brain size
   was significantly decreased in Arhgef2−/− mice ([87]Figure 1A). Nissl
   staining further exhibited reduced thickness of frontal and motor
   cortex in Arhgef2−/− mice ([88]Figure 1B).

Figure 1.

   [89]Figure 1
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   Microcephaly in Arhgef2 knockout mouse model (postnatal 6 weeks)

   (A) The brain weight and size were significantly decreased in
   Arhgef2−/− mice. n = 5–8 per genotype, ∗∗∗p < 0.001, ∗p < 0.05, n.s.:
   no significant difference, unpaired t test.

   (B) Nissl staining of sagittal brain sections. Arhgef2−/− mice showed a
   reduced thickness of frontal and motor cortex region compared with
   wild-type ones, 30 μm thick; scale bar, 2.5 mm, n = 4 per genotype,
   ∗∗p < 0.01, ∗p < 0.05, unpaired t test. Data are represented as mean ±
   S.D.

Defective Arhgef2 led to abnormality of neural differentiation

   Our recent study indicated that, in mouse neocortex, Arhgef2 regulates
   neurogenesis from cortical precursor cells ([91]Ravindran et al.,
   2017). As we expected, the number of NeuN^+ cells significantly
   decreased in Arhgef2−/− mice brain cortex compared with wild-type mice
   (postnatal 8 weeks), and it showed no tendency at all layers
   ([92]Figure 2A). To test whether the genetic removal of Arhgef2 affects
   neurite branching and synapse formation during neural differentiation,
   we examined the morphology of cortical neurites and dendrite spines in
   adult Arhgef2−/− mice. Golgi staining demonstrated that the spine
   density and length were significantly below the level of those of
   wild-type mice, with obviously abnormal morphology of neurites.
   Moreover, mature spines, which are characterized by mushroom-like
   shape, significantly reduced in Arhgef2−/− mice compared with wild-type
   mice ([93]Figures 2B and 2C). We also analyzed the expression of
   presynaptic protein Synaptophysin and postsynaptic protein Psd-95.
   Compared with wild-type mice, expression of Psd-95 decreased in
   Arhgef2−/− mice ([94]Figure 2D), whereas Synaptophysin showed no
   significant change (data not shown).

Figure 2.

   [95]Figure 2
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   Abnormality of neural differentiation in Arhgef2-knockout mice model
   (postnatal 6 weeks)

   (A) The number of NeuN^+ cells decreased significantly in Arhgef2−/−
   mice brain cortex compared with wild-type mice. Red: NeuN. Blue: DAPI.
   WM: white matter. Scale bar, 250 μm, n = 6, ∗∗∗p < 0.001, unpaired t
   test.

   (B) Representative images of Golgi staining. In Arhgef2−/− mice,
   morphology of neurites was abnormal. Scale bar, 250 μm.

   (C) Total spines and mushroom spines significantly reduced in
   Arhgef2−/− mice compared with wild-type mice. The red arrow indicates
   mushroom spines. Scale bar, 5 μm. n = 3 per genotype, ∗∗p < 0.01, ∗p <
   0.05, n.s.: no significant difference, unpaired t test.

   (D) Compared with wild-type mice, expression of Psd-95 decreased
   significantly in Arhgef2−/− mice. n = 4 per genotype, ∗p < 0.05,
   unpaired t test. Data are represented as mean ± S.D.

Genetic deletion of Arhgef2 reduced m^6A level in brain cortex

   The present studies provided strong evidence that m^6A deficiency in
   brain disrupted genes related to neuronal differentiation and impaired
   learning behaviors ([97]Edens et al., 2019; [98]Merkurjev, 2018;
   [99]Koranda et al., 2018). To explore whether deficiency of Arhgef2
   affects m^6A level, we measured total m^6A level of adult mouse brain
   cortex and the expression of writer and eraser genes involved in m^6A
   methylation and demethylation. In Arhgef2−/− mice, total m^6A level
   significantly decreased compared with wild-type mice. Meanwhile, mRNAs
   of methylases Mettl3 and Wtap showed a slight reduction without
   significance, whereas Mettl14, a vital factor controlling neural
   differentiation ([100]Yoon et al., 2017), showed an observable
   downregulation. Demethylases Fto and Alkbh5 showed no observable change
   ([101]Figures 3A–3C and [102]S2A). Furthermore, we found Cebpb and
   Gabpa, transcription factors binding at the transcription start site of
   Mettl14, were significantly decreased in Arhgef2−/− mice compared with
   wild-type mice, whereas Cebpa, Elf1, and Spi1 showed no significant
   change ([103]Weng et al., 2018) ([104]Figures 3D and [105]S2B). In
   parallel, Gene Set Enrichment Analysis (GSEA) of bulk RNA sequencing
   showed that the m^6A-associated genes (writers, erasers, readers,
   Cebpb, and Gabpa) were significantly enriched in the downregulated
   genes of Arhgef2−/− mice ([106]Figure 3E).

Figure 3.

   [107]Figure 3
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   Loss of Arhgef2 reduced m^6A level and led to the enrichment of
   m^6A-tagged mRNAs related to neural differentiation

   (A) Total m^6A level of mouse brain cortex was determined by m^6A RNA
   Methylation Quantification Kit. n = 6, ∗p < 0.05, unpaired t test.

   (B) The mRNA level of Mettl14 examined by qRT-PCR was significantly
   decreased in Arhgef2−/− mice compared with wild-type mice. n = 3 per
   genotype, ∗∗∗p < 0.001, unpaired t test.

   (C) Representative western blot on brain cortex. Compared with
   wild-type mice, the protein expression of Mettl14 reduced in Arhgef2−/−
   mice. n = 4 per genotype, ∗p < 0.05.

   (D) mRNAs of Cebpb and Gabpa, the translation factors of Mettl14,
   reduced significantly in Arhgef2−/− mice compared with wild-type mice.
   n = 3 per genotype (3 measurements for each sample), ∗p < 0.05, ∗∗p <
   0.01, unpaired t test. Data are represented as mean ± SD.

   (E) GSEA showed that the 21 m^6A-associated genes (Mettl3, Mettl14,
   Mettl16, Wtap, Rbm15/15b, Kiaa1429, Fto, Alkbh5, Ythdc1/2, Ythdf1/2/3,
   eIF3, Abcf1, Fmr1, Igf2bp1/2/3, Cebpb, Gabpa) were significantly
   enriched in the downregulated genes of Arhgef2−/− mice. n = 3 per
   genotype, q < 0.05, GSEA software v3.1.0.

   (F) GO analysis of 1,622 downregulated m^6A-tagged genes revealed the
   enrichment for biological process terms related to transcription
   factors, cell communication, neurogenesis, synapse organization, and
   cell cycle. FDR, false discovery rate. Enrichment of KEGG pathway genes
   was also demonstrated.

   (G) Venn diagram showing the overlap of m^6A downregulated mRNAs (p <
   0.05) and neural differentiation-associated genes.

   (H) Coverage plot of m^6A modification of Npdc1 and Cend1 mRNAs.

Loss of Arhgef2 inhibited the translation or nuclear export of m^6A-tagged
Npdc1 and Cend1 mRNAs related to neural differentiation

   To explore how the deficiency of Arhgef2 affects neural differentiation
   by m^6A modification, we performed m^6A immunoprecipitation followed by
   RNA sequencing (m^6A-seq). Similar patterns of the total m^6A
   distribution in wild-type and Arhgef2−/− mice were observed, showing
   that m^6A peaks were abundant in the vicinity of start and stop codons
   ([109]Figure S3A). Loss of Arhgef2 resulted in the downregulation of
   1,622 m^6A-containing mRNAs and upregulation of 1,073 m^6A-containing
   mRNAs. The cumulative distribution fraction of log[2] (fold enrichment)
   demonstrated that m^6A methylation of mRNAs tended to be lower in
   Arhgef2−/− mice than that in wild-type mice ([110]Figure S3B). Gene
   Ontology (GO) and KEGG pathway analysis of the 1,622 m^6A-tagged
   downregulated transcripts illustrated enrichment of genes related to
   cell communication, neurogenesis, neural differentiation, synapse
   organization, and cell cycle ([111]Figures 3F and [112]S3C).
   Interestingly, among 29 mRNAs that were both m^6A significantly
   downregulated and neural differentiation-related, we found two of the
   mRNAs (Npdc1 and Cend1) strongly associated with cell cycle exit and
   terminal neural differentiation ([113]Sansal et al., 2000; [114]Politis
   et al., 2007) ([115]Figures 3G and 3H). As Arhgef2 downregulation keeps
   radial precursors cycling, and thereby inhibits neurogenesis
   ([116]Ravindran et al., 2017), we thus speculated that Npdc1 and Cend1
   played a vital role in this process. Compared with wild-type mice,
   protein level of Npdc1 and Cend1 apparently decreased in Arhgef2−/−
   mice, whereas mRNAs showed no significant change by qRT-PCR
   ([117]Figures 4A–4D), suggesting the positive effect of m^6A
   modification on translation, nuclear export, or stability of both Npdc1
   and Cend1. Interestingly, in Arhgef2−/− mice, whereas polysome
   profiling demonstrated the significant decrease of Npdc1 mRNA rather
   than Cend1 mRNA ([118]Figure 4E), nuclear RNA extraction showed an
   increased expression of Cend1 mRNA, but not Npdc1 mRNA
   ([119]Figure 4F). These results indicated that Arhgef2 mediated the
   translation of Npdc1 mRNA and nuclear export of Cend1 mRNA.

Figure 4.

   [120]Figure 4
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   Loss of Arhgef2 inhibited the translation of m^6A-modified Npdc1 mRNA
   and nuclear export of m^6A-modified Cend1 mRNA

   (A–D) mRNA expression of Npdc1 and Cend1 showed no significant change
   determined by qRT-PCR. Protein levels of Npdc1 and Cend1 apparently
   decreased in Arhgef2−/− mice compared with wild-type ones. n.s.: no
   significant difference. n = 4–6 per genotype, ∗p < 0.05, ∗∗p < 0.01,
   unpaired t test.

   (E) Npdc1 mRNA expression in polysome fractions significantly reduced
   in Arhgef2−/− mice compared with wild-type ones, but Cend1 mRNA showed
   no change. Poor expression of lncRNA (Malat1 and Neat1) in polysome
   fraction indicated the successful extraction of polysome fraction from
   other fractions. n.s.: no significant difference. ∗p < 0.05, ∗∗∗p <
   0.001, unpaired t test. Total: total RNA from brain tissue. Polysome:
   polysome fraction RNA.

   (F) Compared with wild-type mice, the nuclear Cend1 level, but not
   Npdc1, was significantly elevated in Arhgef2−/− mice. U1 was used as
   control gene in nuclei. Only low β-actin expression (~5-fold cytoplasm
   increase over nuclear) represented the successful separation of nuclear
   fractions. ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired t test. n.s.: no
   significant difference. Cyto: cytoplasm. Nuc: Nuclei. Data are
   represented as mean ± S.D.

Overexpression of Mettl14 rescued neurogenesis and morphology of neurons
during neural differentiation

   To characterize the impact of m^6A on neural differentiation, primary
   cells of Arhgef2−/− mice were transfected with HBLV-m-Mettl14/Zsgreen
   ([122]Figure S6). The total m^6A level of primary neurons was
   significantly increased with overexpression of Mettl14
   ([123]Figure S4A). RNA immunoprecipitation (RIP) demonstrated that the
   enrichment of m^6A-tagged Npdc1 and Cend1 mRNAs was significantly
   decreased in Arhgef2−/− RIP samples compared with wild-type RIP
   samples, which could be rescued after transfection with
   HBLV-m-Mettl14/Zsgreen ([124]Figure S4B). Meanwhile, the distinct
   reduction of Npdc1 and Cend1 protein level in primary neurons from
   Arhgef2−/− mice also could be rescued by Mettl14 overexpression
   ([125]Figure 5A). To reveal whether NPCs remain in cell cycle and fail
   to enter neural differentiation pathway, we measured the size of
   cortical neurospheres at 72 h after transfection. A significantly
   increased size of spheres was examined in Arhgef2−/− mice compared with
   wild-type mice, and the phenotypic anomaly could be recovered by
   HBLV-m-Mettl14/Zsgreen ([126]Figure 5B). In addition, we identified a
   fewer number of βIII-tubulin^+ cells (early neuron) and a larger
   proportion of βIII-tubulin^-Ki67^+ proliferating cells (precursors) in
   Arhgef2−/− mice than wild-type ones. Overexpression of Mettl14 was able
   to rescue this phenotype ([127]Figure 5C). There was no significant
   variation in the proportion of phospho-histone H3 (pH3)-positive cells
   within 6 h of 5-ethynyl-2’-deoxyuridine (EdU) exposure (pH3^+EdU^+),
   suggesting that there was no defect in cell cycle to M phase by the
   loss of Arhgef2 ([128]Figure S5). We also found that overexpression of
   Mettl14 rescued impaired neurite outgrowth and post-synaptic formation
   (Psd-95 as marker) in Arhgef2−/− mice ([129]Figures 6A-6C).

Figure 5.

   [130]Figure 5
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   The anomaly of neurogenesis was rescued by overexpression of Mettl14 in
   primary NPCs from Arhgef2−/− mice (at E13.5)

   (A) The protein expression of Npdc1 and Cend1 was examined by western
   blot. n = 3–4 per genotype, ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA.

   (B) The size of cortical neuro-spheres was measured at 72 h after
   transfection. A 2.3-fold increase of sphere size was noted by loss of
   Arhgef2, which could be recovered by HBLV-m-Mettl14-Zsgreen. Green:
   Zsgreen. Scale bar, 100 μm, n = 4 per genotype (3 measurements for each
   group), ∗∗p < 0.01, one-way ANOVA.

   (C) Knockout of Arhgef2 significantly decreased βIII-tubulin^+ cells
   (early neuron) and increased βIII-tubulin^-Ki67^+ proliferating cells
   (precursors), and the proportion could be rescued after Mettl14
   overexpression. Cells were seeded at a density of 5 × 10^4/cm^2 and
   were observed at day 4 after transfection. βIII-tubulin, marker for
   early neuron, green; Ki67, marker for proliferating cells, red; DAPI,
   blue. Scale bar, 50 μm. n = 6 per genotype, ∗∗p < 0.01, ∗∗∗p < 0.001,
   one-way ANOVA. Data are represented as mean ± SD.

Figure 6.

   [132]Figure 6
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   Overexpression of Mettl14 rescued the anomaly of neurite outgrowth and
   post-synaptic formation in primary neurons from Arhgef2−/− mice (at
   E13.5)

   (A) The length of neurites and the number of branches of
   Zsgreen-positive cells was presented by using ImageJ combined with
   NeuronJ and Sholl Analysis plugin. Arhgef2 deficiency reduced the
   longest length of neurites and branch number. Cells were seeded at a
   density of 2.5 × 10^4/cm^2 and were observed at day 3 after
   transfection. Scale bar, 25 μm.

   (B) After transfection with HBLV-m-Mettl14-Zsgreen for 14 days, the
   expression of Psd-95 in primary matured neurons was measured by western
   blot analysis (left). Cells were seeded at a density of 1 × 10^5/cm^2
   and were observed at day 14 after transfection. Data are presented as
   means ± SD from three independent experiments (right), n = 3 per
   genotype, ∗∗p < 0.01, one-way ANOVA. Data are represented as mean ± SD.

   (C) Immunostaining of Psd-95 in primary neurons. Psd-95 (marker for
   post-synapse, red), Zsgreen: green. Scale bar, 2.5 μm.

Overexpression of Npdc1 and Cend1 rescued neurogenesis and morphology of
neurons during neural differentiation

   To further determine the role of Npdc1 and Cend1 in this process, we
   designed to overexpress Npdc1 in Arhgef2−/− mice ([134]Figure S6). The
   measurement of neuro-spheres size suggested that Npdc1 promoted cell
   cycle exit ([135]Figure 7A). An increased number of βIII-tubulin^+
   neurons and decreased number of βIII-tubulin^-Ki67^+ neurons were
   demonstrated in Arhgef2−/− mice transfected with HBLV-m-Npdc1/Zsgreen
   and HBLV-m-Cend1/Zsgreen, respectively ([136]Figure 7B). Meanwhile, the
   abnormal morphology of neurite and expression of Psd-95 could also be,
   to some extent, rescued by overexpression of Npdc1 and Cend1
   ([137]Figures 8A and 8B).

Figure 7.

   [138]Figure 7
   [139]Open in a new tab

   Overexpression of Npdc1 and Cend1 rescued the anomaly of neurogenesis
   in primary NPCs from Arhgef2−/− mice (at E13.5)

   (A) Images of neuro-spheres showing a significant decrease size of
   spheres in Arhgef2-knockout NPCs after transfection with
   HBLV-m-Npdc1/Zsgreen and HBLV-m-Cend1/Zsgreen, respectively. Green:
   Zsgreen. Scale bar, 100 μm, n = 5 per genotype (2 measurements for each
   group), ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA.

   (B) Both Npdc1 and Cend1 promoted the generation of βIII-tubulin^+
   cells (early neuron) and decreased βIII-tubulin^-Ki67^+ proliferating
   cells (precursors). Cells were seeded at a density of 5 × 10^4/cm^2 and
   were observed at day 4 after transfection. βIII-tubulin, marker for
   early neuron, green; Ki67, marker for proliferating cells, red; DAPI,
   blue. Scale bar, 50 μm. n = 6 per genotype, ∗p < 0.05, ∗∗p < 0.01,
   ∗∗∗p < 0.001, one-way ANOVA. Data are represented as mean ± SD.

Figure 8.

   [140]Figure 8
   [141]Open in a new tab

   Overexpression of Npdc1 and Cend1 rescued the anomaly of neurite
   outgrowth in primary neurons from Arhgef2−/− mice (at E13.5)

   (A) Measurement of the length of neurites and the number of branches of
   Zsgreen-positive cells. Cells were seeded at a density of 2.5 ×
   10^4/cm^2 and were observed at day 3 after transfection. Scale bar,
   25 μm.

   (B) After transfection with HBLV-m-Npdc1/Zsgreen and
   HBLV-m-Cend1/Zsgreen for 14 days, respectively, the expression of
   Psd-95 in primary matured neurons was measured by western blot
   analysis. Cells were seeded at a density of 1 × 10^5/cm^2 and were
   observed at day 14 after transfection. n = 4 per genotype, ∗p < 0.05,
   ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA. Data are represented as mean ±
   SD.

Discussion

   m^6A is emerging as a vital factor regulating neural differentiation
   ([142]Yoon et al., 2017; [143]Meyer et al., 2012). Here, we illustrated
   that loss of Arhgef2 reduced m^6A level of brain cortical neurons.
   Specifically, methylases Mettl14 showed the significant reduction,
   suggesting its critical role in neural differentiation regulated by
   Arhgef2. Interestingly, defective Arhgef2 decreased m^6A modification
   of target mRNAs, and thereby regulated not only neurogenesis but also
   neurite outgrowth and synaptic formation during neural differentiation.
   The abnormal phenotypes could be rescued by Mettl14.

   As Arhgef2 down-regulation inhibits neurogenesis by keeping radial
   precursors cycling, and similar cellular phenotype is examined in
   Mettl14 knockout mice ([144]Ravindran et al., 2017; [145]Yoon et al.,
   2017), it strongly led us to focus on two m^6A-modified target mRNAs
   (Npdc1 and Cend1), which were associated with cell cycle exit and
   terminal neural differentiation. Several studies indicate that Npdc1 is
   a neuronal differentiation regulator, which interacts with E2F-1 and
   inhibits cell cycle progression by affecting the functions of E2F-1
   ([146]Sansal et al., 2000; [147]Evrard et al., 2004). Npdc1 positively
   regulates neurite outgrowth and involves in NRF-1-regulated neurite
   outgrowth ([148]Tong et al., 2013). Cend1 (cell cycle exit and neuronal
   differentiation protein 1) coordinates cell cycle exit and
   differentiation of neuronal precursors ([149]Politis et al., 2007).
   Deficiency of Cend1 impairs differentiation of certain types of
   neurons, including Purkinje cells ([150]Sergaki et al., 2010). Both
   Npdc1 and Cend1 are expressed in dividing NPCs and are increased when
   cells start to differentiate. Their expression is developmentally
   regulated and persists in adults along the neuronal lineage
   ([151]Dupont et al., 1998; [152]Gaitanou et al., 2019). Arhgef2
   deficiency decreased m^6A methylations of the Npdc1 mRNA 5′ UTR and
   Cend1 mRNA 3′ UTR, which thus inhibited the translation of Npdc1 and
   nuclear export of Cend1 mRNAs. As overexpression of Mettl14 in primary
   neurons from Arhgef2−/− mice rescued m^6A methylations of both target
   mRNAs, and thereby the protein level of Npdc1 and Cend1 was recovered
   near to wild-type ones, it suggested a significant impact of
   Mettl14-regulated m^6A methylations on the translation of target mRNAs.
   Overexpression of Npdc1 and Cend1 rescued the anomaly of neurogenesis,
   neurite outgrowth, and synaptic formation, indicating their persistent
   critical role during neuronal differentiation.

   Synaptic formation is regarded as a vital process in postnatal brain,
   which is involved in precise communication between neurons and other
   cellular partners. Psd-95, the excitatory post-synaptic marker, has
   been implicated in synaptic development and plasticity, and its defects
   disturb learning and memory ([153]Yoo et al., 2019; [154]Zhang and
   Lisman, 2012; [155]Zheng et al., 2011; [156]Grant, 2012). In our work,
   loss of Arhgef2 led to deficits in spine density and morphology and
   reduced expression of Psd-95 significantly. m^6A sequencing of mouse
   cortex also revealed enrichment of mRNAs related to cell communication
   and synapse organization. Deficits of synaptic formation thus were a
   pivotal factor to explain the intellectual disability caused by Arhgef2
   deficiency.

   Arhgef2 catalyzes the replacement of GDP to GTP on RhoA
   ([157]Birkenfeld et al., 2007). The GTP-bound form of RhoA causes
   Rho-associated kinase (ROCK) activation. Accumulating evidence point
   out that the RhoA/ROCK signaling participates in various neuronal
   functions, such as migration, dendrite development, and axonal
   extension ([158]Fujita, 2014; [159]Rico, 2004). In the last study, we
   reported that ARHGEF2-RhoA/ROCK pathway is essential for the migration
   and specification of precerebellar neurons. Specifically, the
   RhoA/ROCK/MLC pathway is most likely impaired in humans with the
   ARHGEF2 mutation ([160]Ravindran et al., 2017). Several studies have
   indicated the role of RhoA/ROCK related to neural differentiation. NPCs
   with RhoA deficiency exhibit accelerated proliferation and reduction of
   cell cycle exit ([161]Katayama et al., 2011). Activation of the
   RhoA/ROCK/JNK pathway could promote neuronal differentiation ([162]Park
   et al., 2018).

   However, whether ARHGEF2-RhoA/Rock pathway associated with m^6A
   modification during neural differentiation is still unclear. We thus
   examined the expression of Mettl14 in wild-type primary neurons with
   treatment of Rock inhibitor Y27632. Surprisingly, mRNA and protein
   level of Mettl14 showed no significant change after treatment with Rock
   inhibitor, indicating that RhoA/Rock pathway maybe not implicated in
   Arhgef2/Mettl14-induced neuronal differentiation ([163]Figure S7).
   Therefore, the exact pathway by which Arhgef2 regulates Mettl14 is
   worthy to be explored in the future work. Previous studies reported
   that Cebpb, Gabpa, Elf1, Cebpa, and Spi1 have binding sites within 5 kb
   upstream of the transcription start site of Mettl14. Among them, Gabpa
   and Elf1 showed a significant positive correlation, whereas Spil showed
   a significant negative correlation, with Mettl14 ([164]Weng et al.,
   2018). In Arhgef2−/− mice, Cebpb and Gabpa were significantly reduced
   compared with wild-type mice, suggesting Arhgef2 was the most likely
   regulated expression of Mettl14 by them. This work elucidated a
   mechanism by which Arhgef2 deficiency inhibited the translation of
   m^6A-modified Npdc1 and nuclear export of Cend1 mRNAs to impair neural
   differentiation. Intriguingly, it provided a considerable insight to
   think about the biological effects of the GEFs family on brain
   development associated with m^6A modification.

Limitations of the study

   This study linked Arhgef2 and neurogenesis via m6A methylation of
   target mRNAs, including those of Npdc1/Cend1, which are involved in
   cell cycle exit and neuronal differentiation. However, according to our
   present study and others' literatures, it is still hard to know how
   Arhgef2 affects Mettl14 through Cebpb and Gabpa. Arhgef2, a member of
   the Rho guanine nucleotide exchange factor family, is crucial for
   controlling the Ras superfamily. Cebpb is an essential downstream
   mediator of Ras signaling, which could be phosphorylated by Ras
   expression. We thus speculate that Arhgef2 may regulate the expression
   of Cebpb via Ras signaling. As for Gabpa, it is hard for us to
   speculate based on literatures.

STAR★methods

Key resources table

   REAGENT or RESOURCE SOURCE IDENTIFIER
   Antibodies
     __________________________________________________________________

   Mouse monoclonal anti-m^6A Millipore Cat no: MABE1006
   Mouse monoclonal anti-NeuN Millipore Cat no: MAB377B;
   RRID: [165]AB_177621
   Mouse monoclonal anti-β-III tubulin Millipore Cat no: MAB1637;
   RRID: [166]AB_2210524
   Rabbit polyclonal anti-Ki67 Abcam Cat no: 9129;
   RRID: [167]AB_2687446
   Rabbit polyclonal anti-Psd-95 Cell Signaling Technology Cat no:
   3450;RRID:
   AB_2292883
   Mouse monoclonal anti-Synaptophysin Abcam Cat no: ab8049;
   RRID: [168]AB_2198854
   Rabbit polyclonal anti-Mettl14 Cell Signaling Technology Cat no:51104S;
   RRID: [169]AB_2799383
   Rabbit polyclonal anti-Npdc1 Affinity Cat no: DF4225;
   RRID: [170]AB_2836576
   Rabbit monoclonal anti-Cend1 Abcam Cat no: ab113076;
   RRID: [171]AB_10860676
   Rabbit polyclonal anti-Phospho-Histone H3 (Ser10) Cell Signaling
   Technology Cat no:9701;
   RRID: [172]AB_331535
   Rabbit monoclonal anti-β-actin(D6A8) Cell Signaling Technology Cat no:
   8457;
   RRID: [173]AB_10950489
   Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody,
   Alexa Fluor 568 Thermo Fisher Scientific Cat no: A-11036;
   RRID: [174]AB_10563566
   Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody,
   Alexa Fluor 647 Thermo Fisher Scientific Cat no: A-21236;
   RRID: [175]AB_2535805
   Anti-rabbit IgG, HRP-linked Antibody Cell Signaling Technology Cat no:
   7074;
   RRID: [176]AB_2099233
   Anti-mouse IgG, HRP-linked Antibody Cell Signaling Technology Cat no:
   7076;
   RRID: [177]AB_330924
     __________________________________________________________________

   Deposited data
     __________________________________________________________________

   m^6A-sequencing data This paper PRJNA730635
     __________________________________________________________________

   Critical commercial assays
     __________________________________________________________________

   FD Rapid GolgiStain^TM kit Neuro Technologies Cat no: PK401
   m^6A RNA Methylation Quantification Kit (Colorimetric) Abcam Cat no:
   ab185912
   Imprint RNA Immunoprecipitation (RIP) Kit Sigma-Aldrich Cat no:
   RIP-12RXN
   Click-iT EdU Imaging Kits Thermo Fisher Scientific Cat no: [178]C10339
     __________________________________________________________________

   Experimental models: Organisms/strains
     __________________________________________________________________

   Arhgef2 knockout mice Shanghai Biomodel Organism Science & Technology
   Development N/A
     __________________________________________________________________

   Oligonucleotides
     __________________________________________________________________

   β-actin: (F) 5’-GGCTGTATTCCCCTCCATCG-3’
   (R) 5’-CCAGTTGGTAACAATGCCATGT-3’ This paper N/A
   Arhgef2: (F) 5’-ATCTCCGTCTCCGGCATGA-3’
   (R) 5’-CAGGGTGTCTTTACAGCGGTT-3’ This paper N/A
   Npdc1: (F) 5’-CTTCAACACCACGAATCTCGC-3’
   (R) 5’-CCTTTGGAGGTTCCTTATGCC-3’ This paper N/A
   Cend1: (F) 5’-ACCAGCCAAGGCAGATCCT-3’
   (R) 5’-GGTCAAGTTCTCACAAGGCCA-3’ This paper N/A
   Mettl14: (F) 5’-TCTGAGAGTGCGGATAGCATTG-3’
   (R) 5’-GAGCAGATGTATCATAGGAAGCC-3’ This paper N/A
   Mettl3: (F) 5’-CTGGGCACTTGGATTTAAGGAA-3’
   (R) 5’-TGAGAGGTGGTGTAGCAACTT-3’ This paper N/A
   Wtap: (F) 5’-ATGGCACGGGATGAGTTAATTC-3’
   (R) 5’-TTCCCTTAAACCAGTCACATCG-3’ This paper N/A
   Fto: (F) 5’-CCGTCCTGCGATGATGAAGT-3’
   (R) 5’-CCCATGCCGAAATAGGGCTC-3’ This paper N/A
   Alkbh5: (F) 5’-TTGCCACCCAGCTATGCTTC-3’
   (R) 5’-CAGACCGCCGGTTTTCTTCTT-3’ This paper N/A
   Gabpa: (F) 5’-AGCGCATCTCGTTGAAGAAG-3’
   (R) 5’-TCCTGCTCTTTTCTGTAGCCT-3’ This paper N/A
   Cebpb: (F) 5’-CAAGATGCGCAACCTGGAGA-3’
   (R) 5’-GACAGCTGCTCCACCTTCTT-3’ This paper N/A
   Cebpa: (F) 5’-CAAGAACAGCAACGAGTACCG-3’
   (R) 5’-GTCACTGGTCAACTCCAGCAC-3’ This paper N/A
   Elf1: (F) 5’-TGTCCAACAGAACGACCTAGT-3’
   (R) 5’-CACACAAGCTAGACCAGCATAA-3’ This paper N/A
   Spi1: (F) 5’-ATGTTACAGGCGTGCAAAATGG-3’
   (R) 5’-TGATCGCTATGGCTTTCTCCA-3’ This paper N/A
   Malat1: (F)5’-CATGGCGGAATTGCTGGTA-3’
   (R)5’-CGTGCCAACAGCATAGCAGTA-3’ This paper N/A
   Neat1: (F)5’-TGGCCCCTTTTGTTCATTAGC-3’
   (R)5’-TGGAAGGCCATTGTTTCAGG-3’ This paper N/A
   U1: (F) 5’-GATACCATGATCACGAAGGTGGTT-3’
   (R) 5’-CACAAATTATGCAGTCGAGTTTCC-3’ This paper N/A
   Recombinant DNA
   HBLV-m-mettl14-3xflag-Zsgreen-PURO Hanbio N/A
   HBLV-m-Npdc1-3xflag-Zsgreen-PURO Hanbio N/A
   HBLV-m-Cend1-3xflag-Zsgreen-PURO Hanbio N/A
     __________________________________________________________________

   Software and algorithms
     __________________________________________________________________

   Prism 7 GraphPad SCR_002798; [179]https://www.graphpad.com
   ImageJ Wayne Rasband National Institutes of Health
   [180]https://imagej.nih.gov/ij/
   Fastp [181]Chen et al., 2018 [182]https://github.com/OpenGene/fastp
   IGV [183]Meng et al., 2014 [184]http://www.igv.org
   [185]Open in a new tab

Resource availability

Lead contact

   Further information and requests for resources and reagents should be
   directed to and will be fulfilled by the Lead Contact, Hao Hu
   ([186]huh@cougarlab.org).

Materials availability

   This study did not generate new unique reagents.

Data and code availability

   Sequencing data have been deposited with the Sequence Read Archive
   (SRA) under BioProject
   :PRJNA730635([187]https://www.ncbi.nlm.nih.gov/bioproject/PRJNA730635).

Experimental model and subject details

Mice model establishment

   All mice experiments in this work were approved by the institutional
   animal care and use committee in the Guangzhou Medical University
   (registration No. 2019-436, 2019-694). Arhgef2 knockout mice were
   created using CRISPR/Cas9-mediated genome engineering. Exon 2 was
   selected as the target site, and gRNA was designed as following
   sequences: gRNA1: 5’-ACCTACCTGACCCCGGCGACTGG-3’; gRNA2:
   5’-TATTGGAATGTGGCGTAAGCTGG-3’.

   The zygotes co-injected with Cas9 mRNA and gRNA were cultured in medium
   until the two-cell stage, and then were transferred into the oviducts
   of pseudopregnant ICR females. After being identified by genomic PCR
   and Sanger sequencing, the mutant F0 were bred with C57BL/6J males to
   yield heterozygous Arhgef2 mice. Subsequent breeding of the
   heterozygous Arhgef2 mice generated offspring with wild-type and
   knockout littermates for experiments. For animal experiments, Arhgef2
   knockout male and female mice (sex balance) at age of 6-8 weeks were
   used. For primary cell culture, Arhgef2 heterozygous pregnant mice were
   used.

Cell culture

   Primary mouse NPCs were isolated from Arhgef2 wild-type and knockout
   mice embryonic cortices at E13.5 and cultured in Neurobasal medium
   (Gibco, CA, USA) containing 2% B27 (v/v, Gibco), 0.25% Glutamax, and
   25 μM glutamate. Cells were seeded on dishes precoated with
   poly-D-lysine. To measure neuro-spheres, NPCs were cultured in
   ultra-low attachment plates and the size pf spheres was analyzed by
   Image J. The experiments of neurogenesis were performed on
   density-matched NPCs cultures within the first 4 days, while the
   experiments related to synaptic formation were conducted in
   well-matured neurons at day 14. For the overexpression of Mettl14 or
   Npdc1, transfection of HBLV-m-Mettl14/Zsgreen and HBLV-m-Npdc1/Zsgreen
   (at MOI 10) (Hanbio Biotechnology, Shanghai, China) respectively to
   Arhgef2-knockout primary cells was performed. HBLV-Zsgreen was
   transfected to wild-type and Arhgef2 -knockout cells as control.

Method details

Tissue preparation

   Mice at 6 weeks of age were anaesthetized with 50 mg/kg sodium
   pentobarbital. After perfused with 0.9% NaCl and 4% PFA, brains were
   harvested and fixed in 4% PFA overnight, followed with dehydration by
   15% and 30% sucrose. Sagittal brain sections (30 μm thick) were
   prepared using freezing microtome (CM3050S, Leica, Germany).

Nissl staining

   Brain sections were treated with 75% ethanol for 30 s, dH[2]O for 30 s
   and cresyl violet for 2 min. After dehydration with gradient ethanol
   (75%, 95%, 100%), sections were incubated with xylene and mounted using
   neutral resins. The images were taken under microscope.

Immunostaining

   To determine the number of matured neurons in brain cortex, sections
   were permeabilized with 0.3% Triton X-100 and blocked with goat serum
   for 1 h at room temperature, followed by incubation with mouse
   anti-NeuN antibody at 4°C overnight. After washing, HRP-conjugated
   anti-mouse IgG secondary antibody was applied to the sections for 1 h
   at room temperature. Nuclei were stained using
   4,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific).

   To examine the neurogenesis of NPCs, cells were immersed in 4% PFA for
   fixation, 0.3% Triton X-100 for permeabilization, and then blocking.
   Cells were incubated with mouse anti-β-III tubulin antibody and rabbit
   anti-ki67 antibody overnight, followed by secondary antibodies. To
   observe the synaptic formation, Zsgreen-positive cells were co-staining
   with Psd-95 as above. Immunostaining of pH3 in primary cells exposed to
   EdU was also performed as above. Data were obtained from at least three
   independent experiments and analyzed by ImageJ software.

Golgi staining

   Brain tissue prepared for Golgi staining was performed by using FD
   Rapid GolgiStain^TM kit. According to manufacturer’s instruction,
   tissue was immersed in Solution A/B at room temperature for at least 24
   h, followed by Solution C. All procedures should be protected from
   light. Samples embedded in OCT compound were then sectioned coronally
   (30 μm thick). For staining procedure, sections were placed in Solution
   D/E for 10 min. After washing, sections were dehydrated in 50%, 75%,
   and 95% ethanol, followed by 100% ethanol 4 times for 4 min each rinse.
   Sections then were cleared by xylene and mounted. The confocal imaging
   was achieved using a TCS-SP8 LSM confocal imaging system (Leica,
   Wetzlar, Germany).

Quantification of the m^6A

   The m^6A level of brain cortex was measured using m^6A RNA Methylation
   Quantification Kit (Colorimetric). In brief, total RNA of brain cortex
   was isolated using TRIZOL reagent (Invitrogen, Thermo Fisher
   Scientific, USA). 100 -300 ng total RNA per reaction was added into the
   wells filled with 80 μl Binding Solution. After incubation at 37°C for
   90 min, wells were washed with 1X Wash Buffer. m^6A RNA was then
   captured using Diluted Capture Antibody, following by Diluted Detection
   Antibody and Diluted Enhancer Solution. Developer Solution was added to
   each well and incubated for 1-10 min away from light. Signal was
   detected on a microplate reader at 450 nm.

Quantitative real-time PCR (qPCR)

   Total RNA was extracted from the mice brain tissues using TRIZOL
   reagent (Invitrogen, Thermo Fisher Scientific, MA, USA), and
   first-strand cDNA was synthesized with PrimeScript RT reagent
   (Takara-Clonetech, Shiga, Japan). qRT-PCR was performed using SYBR
   Premix Ex TaqII (Takara-Clonetech) and analyzed with Fast Real Time
   System 7500 (Applied Biosystems). Expression levels of each target gene
   were measured relative to β-actin as internal control. The following
   sequences of primers were exhibited in Table ([188]Table S1). The
   experiments were repeated at least three times, each time with
   consistent results.

m^6A-sequencing

   Total RNA was isolated from the cortex samples of the WT and KO mice
   and purified using TRIzol reagent following the manufacturer's
   procedure. The RNA integrity was determined and confirmed using
   Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0 and
   electrophoresis with denaturing agarose gel, respectively. Using
   Dynabeads Oligo (dT) 25-61005 (Thermo Fisher, CA, USA), Poly (A) RNA
   was purified from 50 μg total RNA under two rounds of purification.
   Under 86°C, the poly (A) RNA was fragmented into small pieces using
   Magnesium RNA Fragmentation Module (NEB, cat. e6150, USA) for 7 min.
   m^6A pull-down was performed using m^6A-specific antibody (No. 202003,
   Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl
   and 0.5% Igepal CA-630) for 2 h at 4°C. Next, the IP RNA was
   reverse-transcribed to cDNA, followed by synthesis of U-labeled
   second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209,
   USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher,
   cat. R0133, USA).

   An A-base was then added to the blunt ends of each strand, which was
   prepared for ligation to the indexed adapters. Single- or dual-index
   adapters were ligated to the fragments; size selection was performed
   with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280,
   USA) treatment, the ligated products were amplified with PCR. The
   average insert size for the final cDNA library was 300 ± 50 bp. The
   2 ×150 bp paired-end sequencing (PE150) was performed on an illumina
   Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China).

Bioinformatic analyses of m^6A-seq

   The reads containing adaptor contamination, low quality bases and
   undetermined bases were removed using the fastp software ([189]Chen
   et al., 2018) with default parameter. Sequence quality of IP and Input
   samples was verified by Fastp. The reads were then mapped to the
   reference genome Mus musculus (Version: v99) using HISAT2 ([190]Kim
   et al., 2015). The m^6A peaks with bed or bigwig format that can be
   adapted for visualization on the IGV software ([191]http://www.igv.org)
   were identified by the R package exomePeak ([192]Meng et al., 2014).
   MEME ([193]Bailey et al., 2009) and HOMER ([194]Heinz et al., 2010)
   were used for de novo and known motif finding, followed by localization
   of the motif with respect to peak summit. Called peaks were annotated
   using R package ChIPseeker ([195]Yu et al., 2015). The expression level
   for all mRNAs from input libraries was determined using StringTie
   ([196]Pertea et al., 2015) by calculating FPKM (total exon fragments
   /mapped reads (millions) × exon length (kB)). Gene ontology analysis
   was performed on the 1622 m^6A downregulated genes using GO enrichment
   analysis tool of Omicshare. KEGG pathway analysis was also performed on
   the 1622 m^6A downregulated genes using pathway enrichment analysis
   tool of Omicshare. GO terms and KEGG pathways with FDR corrected p <
   0.05 was considered significant. Protein-protein interaction network
   was obtained from the STRING database ([197]Szklarczyk et al., 2017).
   The interaction network model was generated using Cytoscape 3.6.1
   ([198]Shannon et al., 2003). The lastest gene list was downloaded from
   the BioMart database ([199]https://www.ensembl.org/biomart/martview/)
   where 447 neural differentiation associated genes were identified
   according to their functional annotation and related GO terms.

Neurite outgrowth analysis

   Neurite outgrowth of Zsgreen-positive cells was measured using Image J
   combined with Neuron J and Sholl Analysis plugin. A vertical line from
   the soma to the tip of the longest branch was drawn to defined as the
   length of neurite. the number of branches was examined by counting the
   intersections of neurites and concentric circles. The radius interval
   between circles was 10 μm per step, ranging from 10 μm to the tips of
   the longest branch.

EdU staining

   Primary cells were labeled with 10 μM EdU solution and incubated for
   4 h at 37°C and then fixed with 4% PFA. After permeabilization with
   0.5% TritonX-100, cells were treated with Click-iT EdU Imaging Kits
   according to manufacturer’s protocol.

RNA immunoprecipitation (RIP)

   After transfection of lentivirus for 3 days, m6A modification on Npdc1
   and Cend1 mRNA was detected using Imprint RNA Immunoprecipitation (RIP)
   Kit. In brief, cells (1-2 X 10^6 each reaction) were collected in mild
   lysis buffer supplemented with protease inhibitor cocktail, 1 M DTT and
   40 U/μl ribonuclease inhibitor. After incubation on ice for 15 min, the
   lysis reactions were centrifuged for 10 min at 16,000 g at 4°C, and
   supernatant was collected. 10% of RIP lysate supernatant per reaction
   was labelled as Input. Each reaction was then incubated with 2 μg of
   anti-m^6A antibody-prebinding protein A magnetic beads on a rotator
   overnight. 2 μg of IgG antibody was used for negative control.
   m^6A-tagged mRNA was eluted by IP buffer and purified using TRIZOL
   reagent. SYBR-green based qRT-PCR was performed as described above.

Western blot

   Protein was extracted using 2% SDS containing protease inhibitor
   cocktail and phosphatase inhibitor (Cell Signaling Technology, MA,
   USA). Samples were separated with 12.5% SDS-PAGE and transferred to
   PVDF membranes (Millipore, MA, USA). The membranes were blocked in 5%
   skim milk for 1 h at room temperature and then incubated overnight at
   4°C with the following primary antibodies: rabbit anti-Npdc1 antibody,
   rabbit anti-Cend1 antibody, rabbit anti-Mettl14 antibody, rabbit
   anti-Psd-95 antibody, and mouse anti-Synaptophysin antibody,
   respectively. After washing with TBST, the membranes were incubated
   with secondary antibodies for 1 h at room temperature. Blots were
   visualized by ChemiDocTM MP system (Bio-Rad, USA) and then quantified
   via ImageJ software.

Polysome profiling

   10%~45% sucrose density gradients were prepared using sucrose gradient
   buffer (10 mM HEPES-KOH, 5 mM MgCl2, 150 mM KCl, 200 U/ml RNase
   inhibitor, 1x protease inhibitor cocktail, 100 μg/ml cycloheximide).
   Brain tissue was grinded in a liquid nitrogen-proof container to obtain
   powder. The powder was collected in a modified lysis buffer (10 mM
   HEPES-KOH, 5 mM MgCl2, 150 mM KCl, 0.5 mM DTT, 100 U/ml RNase
   inhibitor, 1x protease inhibitor cocktail, 100 μg/ml cycloheximide,
   0.5% NP-40, 0.05% Triton-X100) and further homogenized using electric
   grinding rod. The homogenate was centrifuged at 20000 rcf for 10 min at
   4°C or reserved for total RNA isolation. The supernatant was layered
   onto sucrose gradient and centrifuged in a SW-40Ti rotor at 36,000 rpm
   for 2.5 h at 4°C and then analyzed using Gradient Fractionator
   (Biocomp). Polysome fraction was collected and RNA was extracted with
   TRIzol LS Reagent (Invitrogen, MA, USA) according to manufacturer’s
   instructions. qRT-PCR was performed as described above. LncRNA (Malat1
   and Neat1) was used as quality control of polysome fraction.

Nuclear RNA extraction

   Homogenization of mice brain tissue with liquid nitrogen grinding, and
   nuclear fractions were prepared using NE-PER Nuclear and Cytoplasmic
   Extraction Kit (Thermo Fisher Scientific) according to the
   manufacturer’s protocol. 100U/ml of RNase inhibitor (Ambon/Fisher) was
   added to the CERI reagent to prevent RNA degradation. After separation
   from the cytoplasmic fraction, the nuclear fraction was washed twice
   with ice PBS (prepared in RNase-free water), and resuspended in an
   appropriate volume of TRIzol reagent (Invitrogen). RNA was purified
   using the TRIzol reagent manufacturer’s protocol. The efficiency of
   separation was determined by measuring nuclear and cytoplasmic RNA
   fractions by qRT-PCR for the ratio of β-actin expression in nuclei and
   cytoplasm. Only nuclear RNA preparations with low β-actin expression
   (~5-fold cytoplasm increase over nuclear) were used for analysis. To
   compare the expression of Npdc1 and Cend1 mRNA in nuclei, U1 was used
   as control gene.

Quantification and statistical analysis

   Statistical analysis was performed using GraphPad Prism8 software. All
   graphs present mean ± S.D. Statistical comparisons of the data were
   carried out by student’s t-test and One-way ANOVA. P<0.05
   was considered statistically significant and significant level is
   indicated as follows: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Specific
   details including the statistic test and the number of experimental
   replicates were demonstrated in figure legends.

Acknowledgments