- Research article
- Open Access
Mettl14-mediated m6A modification is essential for visual function and retinal photoreceptor survival
BMC Biology volume 20, Article number: 140 (2022)
As the most abundant epigenetic modification of eukaryotic mRNA, N6-methyladenosine (m6A) modification has been shown to play a role in mammalian nervous system development and function by regulating mRNA synthesis and degeneration. However, the role of m6A modification in retinal photoreceptors remains unknown.
We generated the first retina-specific Mettl14-knockout mouse models using the Rho-Cre and HRGP-Cre lines and investigated the functions of Mettl14 in retinal rod and cone photoreceptors. Our data showed that loss of Mettl14 in rod cells causes a weakened scotopic photoresponse and rod degeneration. Further study revealed the ectopic accumulation of multiple outer segment (OS) proteins in the inner segment (IS). Deficiency of Mettl14 in cone cells led to the mislocalization of cone opsin proteins and the progressive death of cone cells. Moreover, Mettl14 depletion resulted in drastic decreases in METTL3/WTAP levels and reduced m6A methylation levels. Mechanistically, transcriptomic analyses in combination with MeRIP-seq illustrated that m6A depletion via inactivation of Mettl14 resulted in reduced expression levels of multiple phototransduction- and cilium-associated genes, which subsequently led to compromised ciliogenesis and impaired synthesis and transport of OS-residing proteins in rod cells.
Our data demonstrate that Mettl14 plays an important role in regulating phototransduction and ciliogenesis events and is essential for photoreceptor function and survival, highlighting the importance of m6A modification in visual function.
Retinal photoreceptors mediate vision formation by converting light stimuli from the environment into neural impulses to be sent to the brain. Photoreceptors are classified into two types according to differences in morphology and function: rods for dim light vision and cones for daylight and high-acuity vision. Both rods and cones are further subdivided in an inner segment (IS) and an outer segment (OS). The OS, where the phototransduction machinery resides, contains high concentrations of phototransduction proteins such as the G-protein transducin and visual pigments within membrane-bound discs, which are responsible for detecting light signals and transducing photons into electrical signals through the phototransduction cascade . The IS houses the biosynthetic machinery and energy sources needed to produce and assemble newly synthesized phototransduction proteins and their associated membranes. The OS and IS of photoreceptors are connected by a highly modified primary cilium, also known as the connecting cilium (CC), which is homologous to the transition zone (TZ) of the primary cilium . The CC is a microtubule-based structure consisting of an axoneme with nine doublet microtubules that are nucleated by nine triplet microtubules at the apical surface of the IS, termed the basal body (BB). The axoneme extends into the photoreceptor OS, where it is converted into singlet microtubules, and occupies 60 ~ 100% of the OS length in rods and 100% of the OS length in cones .
In photoreceptors, the CC serves as a conduit to mediate the transport of photopigment molecules, phototransduction proteins, and phospholipid components of the discs from the IS to the OS [2, 4]. Ciliary dysfunction causes disturbances in the trafficking and docking of specific proteins involved in the development and maintenance of the photoreceptor OS, which ultimately leads to photoreceptor degeneration. A number of ciliary defects are caused by dysfunction of the initial ciliogenesis process. In photoreceptors, ciliogenesis proceeds in a stepwise manner: the BB is derived from the mother centriole and docks on the apical membrane through the function of distal appendages (DAPs), initiating outgrowth of the axoneme . This process depends on multiple molecules residing in the centriole/BB. For instance, CEP164, RAB8/CEP290, ODF2, and pericentrin are required for ciliary assembly, while IFT, CEP57, CEP290, CEP97, CEP131, CEP152, and ALMS1 mainly promote cilium elongation ; however, how this process is regulated remains largely unknown. Defects in ciliogenesis and cilium length control in humans have been implicated in a variety of inherited retinal dystrophies (IRDs), which usually present as isolated diseases or as a feature of complex multiorgan syndromic disorders termed ciliopathies, such as Joubert syndrome, Leber congenital amaurosis, Bardet-Biedl syndrome, and Usher syndrome type 2A . These IRDs are genetically heterogeneous and involve mutations in a large number of genes . An increasing number of causative genes of IRDs have been identified, but they can only explain approximately 60% of cases. The precise molecular mechanisms are still largely unknown.
In addition to classical Mendelian inheritance, epigenetic regulation has been found to play a role in the regulation of numerous cellular activities and pathological processes . N6-methyladenosine (m6A), the most abundant modification of eukaryotic mRNA, participates in multiple biological processes by regulating mRNA metabolism, including alternative splicing, stability, nuclear export, degradation, and translation . In mammalian cells, approximately 0.1 to 0.4% of adenines (A) exhibit m6A methylation, with an average of 3 to 5 methylated sites in each mRNA [12, 13]. m6A modifications generally occur in the RRACH (R = G or A, H = A or C or U) motif and mainly reside near stop codons and the 3′UTR of mRNAs [10, 14]. In addition, m6A modification has been shown to be a dynamic and reversible modification that is deposited by a multicomponent complex with methyltransferase-like (METTL)-3/14 and WTAP as the key methyltransferases and erased by the demethylases FTO and ALKBH5 [15,16,17]. During the methylation process, METTL3 acts as the catalytic component of the complex, while METTL14 functions in structural stabilization and RNA substrate recognition [18, 19]. After transcripts are transported into the cytoplasm, their m6A sites are recognized by a series of m6A readers, such as YTHDF1/2/3, IGF2BP1/2/3 and YTHDC1/2, which then exert regulatory effects [20,21,22,23,24].
New m6A methylation-associated enzymes are frequently identified, and they have been proven to play crucial roles in many essential biological processes, such as cell fate decisions, embryonic development, sex determination, spermatogenesis, tumorigenesis, and T cell homeostasis . In particular, m6A methylation has a wide range of effects on the nervous system and plays important roles in the self-renewal of neural stem cells, learning memory, brain development, and synaptic growth. For instance, specific depletion of m6A in the nervous system by conditional knockout of Mettl14 causes compromised cortical neurogenesis in embryonic mice by regulating the decay of neurogenesis-related transcripts . Analogously, selective inactivation of METTL3 in the mouse nervous system causes severe developmental defects in the brain by controlling the mRNA stability of genes involved in cerebellar development and apoptosis . In addition, deficiency of the m6A eraser FTO affects neurogenesis and hippocampal-dependent memory in mice . In vitro data further indicate that FTO is enriched in axons, and specifically silencing axonal FTO inhibits axon elongation . Furthermore, deficiency of another m6A eraser, ALKBH5, was reported to affect the RNA metabolism of a subset of cell fate determination genes and thus cause defective postnatal cerebellar development . In addition, Ythdf1-deficient mice present diminished learning memory ability with impaired hippocampal synaptic transmission . The proliferation and differentiation of neural stem cells are severely affected by embryonic deletion of Ythdf2 . Very recently, the newly found reader Prrc2a was also proven to maintain normal cognition by determining the specification and myelination of oligodendrocytes . All these studies indicate the important roles of epitranscriptomic m6A modifications in the regulation of neuronal development and neurogenesis. However, the in vivo functions and mechanisms of m6A in the neural retina remain to be characterized. Therefore, we focused on exploring the potential role of m6A in retinal photoreceptor cells.
In the present study, we generated the first retina-specific Mettl14-knockout mice using the Rho-Cre and HRGP-Cre lines and investigated the functions of Mettl14 in retinal rod and cone photoreceptors. Our data showed that loss of METTL14 in rod cells causes a weakened scotopic photoresponse and rod degeneration. Further study revealed the ectopic accumulation of multiple OS proteins in the IS. Deficiency of METTL14 in cone cells led to the mislocalization of cone opsin protein and the death of cone cells. Moreover, Mettl14 depletion resulted in drastic decreases in METTL3/WTAP levels and reduced m6A methylation levels. Mechanistically, transcriptomic analyses in combination with MeRIP-seq illustrated that m6A depletion via inactivation of METTL14 resulted in reduced expression levels of multiple phototransduction- and cilium-associated genes, which subsequently led to reduced levels of OS-resident molecules and compromised ciliogenesis in rod cells. Collectively, our data demonstrated that METTL14 plays an important role in regulating phototransduction and ciliogenesis events and is necessary for photoreceptor function and survival, highlighting the importance of m6A modification in the retina.
Establishment of a specific knockout model of Mettl14 in retinal rod and cone cells
As a key methyltransferase, METTL14 plays important roles in vivo by mediating m6A methylation of mRNA. However, the molecular functions of METTL14 in the retina remain elusive. To determine the role of METTL14 in the retina, we began by examining the expression pattern of Mettl14 in rod and cone photoreceptors. Western blot data first confirmed that the METTL14 protein was abundant in mouse retinas (Additional file 1: Fig. S1A). Immunofluorescence staining of frozen murine retinal sections revealed that METTL14 was widely expressed in whole retina, and distinctly expressed in the IS and outer nuclear layer (ONL) (Additional file 1: Fig. S1B). In ONL, interestingly, the METTL14 immunostaining was most intense near the nuclear periphery of photoreceptor cells (Additional file 1: Fig. S1B), the region where chromatin appears to be least condensed , implying that METTL14 plays a role in m6A modification in photoreceptors.
To investigate METTL14 expression in cone cells, a cone-specific ROSA26-tdTomato reporter line was introduced to monitor the entire cell body of cones (see details in methods), permitting double-labeling analysis with nucleus-localized METTL14. As expected, the immunofluorescence data showed that METTL14 was analogously expressed in the nuclear periphery of tdTomato-expressing cones (Additional file 1: Fig. S1C). Thus, the intense expression of METTL14 in both rod and cone photoreceptors suggests that METTL14 may be involved in the maintenance of photoreceptor cells.
To investigate the potential functional roles of METTL14 in these cells, we generated mice with rod cell-specific METTL14 ablation by crossing Mettl14 [exons 2 to 10] floxed mice (Mettl14flox/flox) with a Rho-Cre driver line in which Cre is targeted to the Rho locus; thus, Cre expression is under the control of the endogenous rod Rho promoter  (Additional file 1: Fig. S1D). In addition, a METTL14 cone knockout line was constructed using HRGP-Cre mice in which Cre is driven by the human red/green pigment gene promoter  (Additional file 1: Fig. S1D). Genotyping results were used to distinguish the littermates of the progeny: Mettl14flox/flox; Rho-Cre (hereafter RKO), Mettl14flox/flox; HRGP-Cre (hereafter HKO), and Mettl14flox/flox (used as control, hereafter Ctrl) (Additional file 1: Fig. S1E).
Specific depletion of Mettl14 in retinal rods causes impaired visual function
To define the retinal phenotype of the constructed RKO mice, we first confirmed the excision efficiency in rods. Western blot analysis indicated that the METTL14 protein level in the retina in RKO mice was reduced to 53% of that in their control littermates (Fig. 1A). Considering that rod cells account for 97% of photoreceptors but ~ 70% of all retinal cells , the deletion efficiency was fairly robust. Immunostaining results further verified that the RKO mice failed to produce the METTL14 protein in the retinal ONL, although METTL14 was still expressed in cones (Fig. 1B).
We next evaluated the physiological status of the RKO retina. Photopic and scotopic electroretinograms (ERGs) of control and RKO mice at 5 months of age were performed. Scotopic ERG recordings, which detect the responses of rod-driven circuits, revealed reduced amplitudes of both a-waves and b-waves compared with those of the controls at each flash intensity (Fig. 1C). The mean amplitudes of the a- and b-waves of RKO mice were reduced by approximately 73% and 67%, respectively (Fig. 1D, E). In contrast, photopic ERG traces, which reflect the status of cone cells, appeared to be normal in RKO mice (Additional file 1: Fig. S2). These results suggested that the rod photoreception function is significantly compromised by the loss of METTL14.
Rod cell degeneration and mislocalization of OS proteins in the RKO retina
To determine the pathological changes underlying the abnormal ERG results, histologic analysis was performed to examine the retinal morphology in RKO retinas at different ages. At 2 months, no obvious difference was observed between the RKO and control mice (Additional file 1: Fig. S3). However, at 3.5 months of age, a shorter OS and thinning of the outer retina were observed in the RKO retina (Fig. 2A). This phenotype worsened at 5 months of age, and the ONL was much thinner, with a 36.6% decrease in thickness (Fig. 2B). At 11-month-old, surprisingly, no OS was left, and the ONL was reduced to 1–2 cell per row in RKO retina (Additional file 1: Fig. S4A). Only trace tissues remained in ONL in 12-month-old RKO mice (Additional file 1: Fig. S4B). In addition, heterozygous Mettl14loxP/+, Rho-Cre mice were tested. The heterozygous RKO mice showed a mild degeneration phenotype characterized by a slightly thinner ONL thickness at 9 months of age, while the ONL of their RKO littermates only had 2–3 cell per row at this stage (Additional file 1: Fig. S5). We thus focused on homozygous RKO mice in the following analysis.
To characterize the ultrastructural features of the photoreceptor OS, electron microscopy was performed and revealed severely disorganized outer segment discs of 3.5-month-old animals. In control siblings, photoreceptor OSs were elongated and contained well organized stacks of discs, generating a palisade pattern on the retinal pigment epithelium (RPE). The OS discs, however, were severely disorganized and fragmented in RKO photoreceptor cells (Fig. 2C), indicating the degeneration and compromised maintenance of the OS.
The OS houses large amounts of phototransduction proteins, and all OS proteins are synthesized in the IS before being transported to the OS across the photoreceptor cilium. To investigate the molecular mechanisms underlying these severe phenotypes, we examined the subcellular localizations of multiple key OS proteins in RKO retinas by immunofluorescence staining. Rhodopsin, the key light receptor in rods that initiates scotopic vision, constitutes over 80% of protein in the disk membrane in the OS of rod photoreceptors . At 3.5 months of age, rhodopsin was well organized and strictly restricted to the rod OSs of control animals, and it was absent in the NaK pump antibody-labeled IS (Fig. 2D). In RKO mutants, however, reduced rhodopsin content was observed, together with punctate structures in which rhodopsin was mislocalized to the IS (Fig. 2D). Analogously, we found that the distribution of the OS structural protein peripherin 2 (PRPH2) displayed similar ectopic IS accumulation in RKO retinas (Fig. 2E). Furthermore, the normal localization of both phosphodiesterase-6-β (PDE6B) and CNGA1, the central phototransduction components, was in the OS in the control retina. In contrast, in the RKO retina, increased PDE6B and CNGA1 immunoreactivity was detected in the IS (Fig. 2F). These results demonstrate that METTL14 deficiency impairs the protein transport of several integral phototransduction pathway proteins in rod photoreceptors, which likely contributes to photoreceptor dysfunction and retinal degeneration.
In addition, immunostaining of retinal sections revealed that the expression of glial fibrillary acidic protein (GFAP) was dramatically upregulated in degenerating retinas compared to control retinas (Additional file 1: Fig. S6A). Western blot analysis further confirmed this result, as revealed by the increased GFAP protein level in the RKO retina (Additional file 1: Fig. S6B, C). These results suggested that Müller cells were activated to proliferate as an indicator of retinal damage . TUNEL analysis revealed several TUNEL-positive nuclei in the ONL of retinas from 5-month-old RKO mice (Additional file 1: Fig. S6D, E), suggesting that the gradual loss of photoreceptors in the RKO retina mainly occurs via apoptosis. Together, these data indicate that ablation of METTL14 results in rod photoreceptor degeneration.
METTL14 depletion in cone cells leads to cone photoreceptor defects
Cone cells are responsible for daylight, central, high-acuity and color vision. We next determined the effect of METTL14 deficiency on cone cells using the constructed HKO mice. METTL14 depletion in cone cells of HKO animals was confirmed. Given that cones constitute only ~ 3% of retinal photoreceptor cells  and using western blotting to confirm the deletion efficiency should be problematic, we introduced ROSA26-tdTomato reporter mice to visualize the specific expression of HGRP-Cre in cone photoreceptors (see details in the Methods). Cone cells were labeled with an antibody specific to cone arrestin (cArr), and HRGP-Cre was distinctly expressed in cArr-marked cone cells (Additional file 1: Fig. S7A), suggesting precise excision. Immunofluorescence staining further verified specific METTL14 depletion in cone cells (Additional file 1: Fig. S7B). Photopic ERG analysis revealed decreased ERG responses in 6-month-old HKO mice in comparison to control (Additional file 1: Fig. S8A, B, E), while no significant difference of scotopic a-wave or b-wave was observed between those two groups (Additional file 1: Fig. S8C, D, F), suggesting impaired cone function. We then double-immunolabeled retinal cryosections from 6-month-old control and HKO mice with cArr as well as Alexa Fluor-594-conjugated PNA, which mainly labels the cone matrix sheaths associated with all three types of cones. The immunofluorescence results revealed a dramatic reduction in cone number in HKO mice (Fig. 3A, B), with a shortened, disorganized, and misshapen cone OS compared to that of controls, suggesting that METTL14 depletion results in the degeneration of cone photoreceptors.
To further examine the pathological changes of cones in HKO mice, retinal flat mounts from young (5-month-old) animals were prepared and coimmunostained with an L/M-opsin antibody to mark the cone OS and Alexa Fluor-594-conjugated PNA. Given that the M-opsin-expressing cones were largely concentrated in the dorsal retina, with very few M-opsin-positive cones in the ventral retina , we quantified the OS length and the number of M-cones in the dorsal retina of HKO and control mice. As expected, a dramatic decrease in the number of M-cone cells was observed in the HKO retina (Fig. 3C, D), in line with the immunolabeling results of cArr. In addition, although the M-opsin-marked OS length was comparable across the different groups during this time, the overall M-opsin fluorescence intensity in HKO retinas was significantly decreased (Fig. 3E), suggesting opsin degradation. Notably, close examination of retina cross-sections in higher-magnification images further revealed the extensive accumulation of M-opsin in the IS and cell body, accompanied by a malformed OS (Fig. 3F). Considering the similar mislocalization pattern of OS proteins observed in RKO rods (Fig. 2E-G), we suggest that METTL14 deficiency-induced defects in protein transport to the OS underlie the degeneration of retinal photoreceptors.
METTL14 deletion decreases the stability of m6A methyltransferases and the abundance of m6A methylation in the RKO retina
METTL14 is known to physically and functionally interact with the RNA methyltransferase METTL3 and the RNA binding protein WTAP to constitute the core components of the RNA methyltransferase complex . In addition, METTL14 and METTL3 were reported to regulate each other’s stability at the protein level . These results prompted us to examine the expression level and cellular distribution of METLL3 and WTAP in METTL14-deficient RKO retinas. Western blot data showed that the expression level of METTL3 was reduced to 37% of that of controls, while the WTAP protein level in the RKO retina was reduced to 68% of that of controls (Fig. 4A, B). Consistent with this finding, using immunofluorescence microscopy, we found that METTL14 depletion decreased the expression of both METTL3 and WTAP to varying degrees in the ONL of RKO retinas, with METTL3 almost absent in rod cells, while WTAP was still present in the ONL but showed a lower content (Fig. 4C, D). In contrast, the remaining METTL3/WTAP signal was still expressed in cone cells of RKO retinas (Fig. 4C, D, arrows). These findings suggested that METTL14 depletion leads to impaired stability of the m6A methyltransferase complex. To investigate whether the altered protein level affects METTL14-mediated m6A methylation, the global profiles of m6A in RKO retinas were detected by dot blot analysis. As expected, there was a 40 to 50% reduction in overall m6A levels in the RKO retina compared with the control retina (Fig. 4E, F). Taken together, these results suggest that METTL14 is critical for the stability of the m6A methyltransferase complex and m6A methylation in retinal rod cells.
Transcriptome-wide MeRIP-seq and RNA-seq assays to identify potential targets of METTL14
To investigate the variations in m6A modification of specific genes, we performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) on 3-month-old control and RKO retinas, with independent biological replicates. De novo motif analysis using HOMER identified “GGACU” as the most common m6A motif in m6A-immunoprecipitated RNAs from control retinas, while the m6A peaks in RNAs from RKO retinas were enriched in the regions with GGACR motifs (Fig. 5A), in accordance with observations in the mammalian system [20, 43].
In total, m6A-seq identified 24,044 and 22,405 m6A peaks from 8402 and 7777 m6A-modified transcripts in control and RKO retinas, respectively (Fig. 5B). In the RKO retina, 5329 peaks disappeared and 4023 peaks appeared, while the remaining 18,382 peaks were found in both RKO and control retinas (Fig. 5B). Regarding m6A-modified genes, m6A-seq identified 794 newly modified genes and 1234 genes showing loss of m6A modification in the RKO retina, while the other 6983 genes were found in both control and RKO retinas (Fig. 5C). Given that METTL14 is a key component of the m6A methyltransferase complex, these data indicated that the identified unique peaks and genes are supposed to contain genuine targets of METTL14. We further analyzed the density distribution of m6A peaks across the mRNA transcripts, and they were found to be predominantly localized in the stop codon, covering the CDS and 3′ UTR in both control and RKO retinas, although fewer m6A peaks were enriched near the stop codons in the RKO retina (Fig. 5D, E).
To explore how METTL14 depletion affects m6A-containing transcripts, we next employed RNA sequencing (RNA-seq) to identify the affected genes in control and RKO retinas. Considering that rods occupy only the outermost layers of the neurosensory retina, genes with fold change (FC) > 1.2 and adjusted p-value < 0.05 were defined as differentially expressed genes. Through analysis of our RNA-seq data, we demonstrated that the mRNA transcript levels of 932 genes were upregulated and those of 2002 genes were downregulated in RKO retinas compared to control retinas (Fig. 5F). We filtered these 2,002 downregulated genes that were found by RNA-seq with 1,234 genes that lost their m6A peaks in the RKO retinas, which resulted in the identification of 224 candidate genes. We next focused on these 224 genes to obtain insights into the molecular mechanisms (Fig. 5G). Gene ontology (GO) pathway analysis of these 224 genes indicated that a set of genes were particularly associated with phototransduction (Fig. 5H). Moreover, analysis of cellular components further revealed that m6A modifications were enriched in a handful of genes related to the photoreceptor cilium (Fig. 5I). All enriched GO terms in terms of biological processes and cellular component are shown in Additional file 2 and 3, respectively. Interestingly, the phototransduction cascade initiates visual processing in the photoreceptor OS, which is actually derived from a highly specialized primary cilium termed the connecting cilium. Ciliary dysfunction generally causes compromised visual processing, which in turn induces degeneration of mature photoreceptors. Therefore, we focused on the phototransduction process and the photoreceptor cilium in the following investigation.
METTL14 regulates the m6A modification and translation of multiple phototransduction-related genes in rods
Light absorption and phototransduction by photoreceptors represent the initial steps of vision, and the unveiled phototransduction pathway prompted us to further explore the target genes of METTL14. Heatmaps of the RNA-seq results showed that the mRNA expression of multiple phototransduction-related genes (Rho, Gnat1, Guca1b, Pde6b, Reep6, Rgs9bp, Rs1, Prph2, Unc119, and Rgs9) was significantly downregulated in the RKO retina (Fig. 6A). RT-qPCR further confirmed the downregulation of the genes Rho, Gnat1, Guca1b, Pde6b, Prph2, and Unc119 in the RKO retina (Fig. 6B).
We then asked whether the altered gene expression caused by METTL14 depletion was a consequence of suppressed m6A methylation. We first compared the m6A peaks of these altered transcripts between control and RKO retinas using the Integrative Genomics Viewer (IGV) tool, which revealed that the m6A sites of the Rho, Gnat1, Guca1b, Pde6b, Prph2, and Unc119 transcripts were mainly distributed in start codons, in exons and around stop codons (Fig. 6C, see details in Additional file 4: Table S1). Moreover, METTL14 silencing broadly reduced the m6A methylation abundance in Rho, Gnat1, Guca1b, Pde6b, Prph2, and Unc119 in RKO retinas compared to controls (Fig. 6C). Several significantly (FC > 1.5, p < 0.05) decreased m6A methylation sites of these transcripts were filtered out and validated by MeRIP-qPCR with specific primers in fragmented mRNA samples (Additional file 4: Table S1), which confirmed the reduced enrichment of m6A at mapped sites in Rho, Gnat1, Guca1b, Pde6b, Prph2, and Unc119 (Fig. 6D).
In addition, when we examined the expression of the corresponding proteins by western blotting, we observed a reduction in RHO, GNAT1, PDE6B, and PRPH2 protein levels in the RKO retina (Fig. 6E, F), suggesting that deletion of METTL14 might decrease the m6A levels of these phototransduction-related gene transcripts, ultimately leading to altered expression. Interestingly, however, another key component in phototransduction, GRK1 (which showed no significant differences in RNA or m6A levels in the RKO retina), appeared to have protein levels comparable to those of control littermates (Fig. 6E, F), indicating that METTL14-mediated m6A modification should be selective.
Together, these results demonstrate that a subset of phototransduction-associated genes are downregulated under METTL14-silenced conditions, highlighting the indispensable role of METTL14-mediated m6A modification in rod phototransduction processes.
m6A depletion suppresses ciliogenesis by reducing the expression of ciliu-associated genes
Given that the genes that encode components of the cilium were also highly enriched, we next explored the roles of METTL14 and its corresponding m6A modification on the photoreceptor cilium. As mentioned above, a subset of downregulated cilium-associated genes with m6A loss were unveiled in the RKO retina, including Cep164, Fam161a, Cep250, Crocc, Cc2d2a, Arl3, and Mast2. To validate the sequencing results, we first performed RT-qPCR, and our results showed that Cep164, Fam161a, Arl3 and Mast2 were significantly downregulated in METTL14 RKO retinas (Fig. 7A).
Subsequently, to investigate the impact of METTL14 depletion on the m6A modification of these filtered genes, the m6A peak distribution was monitored using IGV tools. The mRNAs of the indicated cilium-related genes were found to have multiple m6A peaks near the start codon, stop codon, and deposited broadly across multiple exons (Fig. 7B, see details in Additional file 4: Table S1). To better quantify the m6A abundance of these methylated sites in control and RKO retinas, we performed a MeRIP-qPCR assay and detected significant decreases in the enrichment of Cep164, Fam161a, Arl3, and Mast2 m6A levels in RKO retinas (Fig. 7C), further confirming that our MeRIP-seq data were robust and accurate.
The above findings suggest that METTL14 depletion could reduce the expression of cilium-associated genes. Indeed, among these indicated genes, CEP164 is a centriole DAP protein required for ciliogenesis [44, 45]. FAM161A and ARL3, likewise, were proven to be required for cilium assembly [46,47,48]. MAST2 is associated with microtubules, although its role in the retina remains unknown [49, 50]. These results remind us that METTL14 presumably maintains the function and survival of photoreceptor cells by regulating the process of cilium formation and assembly. To verify this hypothesis, cryosections of retinas from 3-month-old control and RKO animals were prepared and subjected to immunofluorescence staining with antibodies against the DAP marker CEP164 and the ciliary marker acetylated α-tubulin (Ac-tubulin). Strikingly, photoreceptors in the control retina exhibited elongated and aligned cilia, while RKO photoreceptors presented short, irregular and scattered cilia with a disordered orientation (Fig. 7D). Specifically, the mean cilium lengths were significantly shorter in RKO photoreceptors (Fig. 7E, 1.17 ± 0.08 μm, n = 24) than in control photoreceptors (0.53 ± 0.12 μm, n = 18), suggesting impaired ciliary assembly. In addition, quantitation of CEP164-marked DAPs revealed a 26.3% decrease in RKO retinas compared to control retinas (Fig. 7F). DAPs, localized in the distal end of the basal body are required for BB docking and subsequent ciliogenesis. Thus, combined with the RNA-seq and RT-qPCR results, it was preliminarily determined that cilium dysfunction in RKO retinas mainly resulted from reduced CEP164 levels and corresponding DAP loss, which ultimately prevented cilium initiation. Consistently, the western blot results further showed that the Ac-tubulin content was clearly attenuated in RKO retinas compared to control retinas (Fig. 7G), confirming the reduced cilium abundance caused by defects in ciliogenesis.
To observe ultrastructural abnormalities, an electron microscope assay was employed and further showed that the absence of METTL14 significantly affected ciliogenesis or cilium assembly in rod cells (Fig. 7H). Although the overall morphology and relative positioning of accessory ciliary structures appeared normal, there was a decrease in the length of the CC and BB by at least 50% compared with that of the age-matched control mice (Fig. 7H, I), consistent with Ac-tubulin immunolabeling. In particular, the CC average length was dramatically decreased in 3-month-old RKO photoreceptors (1.37 ± 0.05 μm) compared with control photoreceptors (0.68 ± 0.06 μm), while the BB in RKO tissues had a slightly reduced length (Fig. 7H, I), suggesting that the BB may indeed fail to undergo complete maturation and subsequent elongation. The CC of photoreceptor cells plays a crucial role in the selective transport of proteins and other molecules to and from the OS. Based on the above mentioned mislocalization of phototransduction proteins in both RKO and HKO retinas, our study demonstrated that Mettl14 deficiency impairs photoreceptor ciliogenesis by decreasing expression of associated ciliary proteins, which in turn induces ectopic accumulation of OS proteins and ultimately photoreceptor degeneration.
Among posttranscriptional modifications, m6A methylation is the most abundant mammalian internal mRNA modification, and it plays a central role in translational control. However, the necessity of m6A tagging for the normal function of retinal photoreceptor cells has not been definitively demonstrated. In the present study, by using cell type-specific conditional depletion of Mettl14 in either rod or cone photoreceptors, we show that METTL14-catalyzed m6A modification is indispensable for the function and survival of both rods and cones, likely via the translational regulation of phototransduction- and ciliogenesis-associated genes, which consequently leads to the downregulation and ectopic accumulation of multiple phototransduction molecules residing in the OS and ultimately induces photoreceptor degeneration. Our findings demonstrated, for the first time, the regulatory function of m6A modification in mammalian retinal photoreceptors.
Using high-throughput MeRIP-seq and RNA-seq, we investigated the regulation of METTL14-mediated m6A modification in photoreceptors and revealed two potential mechanisms underlying retinopathy in RKO mice. One potential mechanism involves altered expression of phototransduction-related genes, as numerous key molecules involved in the phototransduction cascade and associated structural OS proteins are modified by m6A, directly affecting the visual impulses of rod photoreceptors. Here, we demonstrated that the transcripts of several phototransduction signaling genes, including the key components in the RHO-Transductin-PDE6 pathway (Rho, Gnat1, Pde6b, Guca1b, Unc119) and the structural OS protein-encoding gene Prph2, present lower levels of m6A methylation (Fig. 6A, B). In addition, a reduction in RHO, GNAT1, PDE6B, and PRPH2 protein levels was detected in the RKO retina (Fig. 6E, F), which likely contributes to the dysfunction of rod cells. It is unlikely that reduced phototransduction protein levels in the RKO retina were the cause of rapid photoreceptor cell degeneration, as GRK1, another key phototransduction protein located in the OS, was present at levels comparable to those of control littermates during this period (Fig. 6E, F), suggesting that the expression changes of these genes are mainly caused by METTL14 depletion-induced m6A loss rather than photoreceptor degeneration. Surprisingly, all of the identified genes play key roles in light transduction, and deletion/mutation of these genes could lead to severe retinal denegation in humans and mice. For instance, mutations in Rho are a frequent cause of retinitis pigmentosa (RP) and less often congenital stationary night blindness (CSNB) . The gene Gnat1, which encodes the transducin alpha subunit, has also been implicated in CSNB  and late-onset rod-cone dystrophy . Pde6b, encoding the catalytic beta subunit of PDE6, was one of the first genes identified as causing autosomal-recessive RP and CSNB in humans , and Pde6b mutant mice (Pde6brd1 and Pde6brd10) exhibit rapid photoreceptor degeneration, diminished ERG responses, and abnormal OS morphology . Guca1b, which encodes guanylyl cyclase activating protein 2 (GCAP2), a calcium-binding protein that activates photoreceptor GCs, is also associated with RP in humans . In addition, UNC119, which was originally identified in a screen for candidate retinal degeneration genes , predominates in the photoreceptor IS and synaptic regions and is essential for transducin trafficking toward the OS . Apart from these, PRPH2 is a photoreceptor-specific glycoprotein necessary for the stabilization and compaction of OS disks, and mutations of PRPH2 have also been associated with RP and macular/pattern dystrophies . PRPH2-deficient (rds/rds) mice fail to develop OSs and undergo subsequent retinal degeneration . As stated above, all of these identified genes play vital roles in photoreceptors and are associated with retinopathy. It makes sense that the downregulation of these genes in the RKO retina contributes to photoreceptor dysfunction and degeneration.
In addition, we also filtered out four molecules in the ciliary pathway, Cep164, Fam161a, Arl3, and Mast2, which are crucial for the early ciliogenesis steps (BB migration and docking) as well as the subsequent assembly process. Specifically, CEP164, a key component of DAPs, has been proven to be required for BB docking and ciliogenesis. In humans, recessive mutations of CEP164 are associated with nephronophthisis and retinal degeneration . Mice with germline knockouts of CEP164 present defects in BB docking to cell membranes, thereby generating syndromic ciliopathies . Additionally, disruption of CEP164 in RPE cells blocked primary cilium formation . Consistent with these studies, our results indicate a 26.3% decrease in CEP164-marked DAPs in the RKO retina (Fig. 7D, E), which consequently presented fewer and disordered CCs, suggesting dysfunction of ciliogenesis. Another gene identified in this study was Arl3, which encodes a protein essential for elongation of tubules; deletion of this gene could prevent formation of an axoneme. In particular, the BBs in Arl3−/− photoreceptors could dock to the cell membrane without CCs, suggesting that ARL3 does not affect BB docking but prevents the formation of axonemes. Since we detected reduced m6A content and of Arl3 expression levels in the RKO retina and further showed that the cilium length in RKO retinas was decreased (Fig. 7D, F), it is reasonable to speculate that the absence of Mettl14 affects not only ciliogenesis but also cilium assembly. In addition, we revealed the downregulation of Fam161a, which encodes Fam161a, which is localized to the base of the CC, the BB region, and the adjacent centriole in photoreceptor cells . Mutations in Fam161a are the most common cause of autosomal recessive retinitis pigmentosa in the Israeli-Jewish population , implying the vital roles of this protein in RP pathologies. Moreover, MAST2, which is a microtubule-associated protein essential for cell division, survival and tumorigenesis, was also identified here . However, until now, little has been known about the role of MAST2 in the retina. Our study suggests that it likely plays a role in photoreceptors, thereby providing clues for further investigation.
Notably, ciliary defects in photoreceptors usually result in aberrant OS protein accumulation and are associated with several forms of inherited retinal degeneration . Analogously, we showed that multiple OS proteins cannot be properly translocated and are mislocalized in the IS in both Mettl14-deficient rods (Fig. 2D- F) and cones (Fig. 3F), mainly due to impaired cilia biogenesis. Protein aggregation disrupts photoreceptor function and produces cytotoxic effects, which ultimately accelerate cell death. Indeed, abnormal ciliogenesis and blunted ciliary trafficking are major causes of a wide variety of clinical manifestations commonly referred to as ciliopathies , yet the pathogenic mechanisms remain largely unknown. We, for the first time, demonstrated that m6A methylation has a regulatory effect on cilium biogenesis, thereby offering a new viewpoint to elucidate the mechanism of related ciliopathies.
Our study is the first to demonstrate that m6A is critical for maintaining the function and survival of retinal photoreceptors. We reported that METTL14 deficiency in retinal photoreceptors induces attenuated m6A methylation followed by transcriptome-wide gene expression dysregulation. We provide evidence that METTL14 inhibition suppresses retinal phototransduction and ciliogenesis processes by decreasing the expression of specific phototransduction- and cilium-related genes. Considering the critical role of phototransduction and ciliary pathways in RP retinopathy, this study provides novel insights for RP therapeutic intervention.
All animal experiment protocols were approved by the Institutional Animal Care and Use Committee of Sichuan Provincial People’s Hospital (Chengdu, Sichuan, China) and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All animals were housed in a temperature-controlled room (24 °C) with a 12-h light/dark cycle. Mice were maintained on a C57BL/6 J background.
Mice with a Mettl14 deletion specifically in retinal rod or cone cells were generated using the Cre-loxP system. Briefly, Mettl14loxP/loxP mice  were crossed with B6.Cg-Pde6b < + > Tg(Rho-icre)1Ck/Boc (Jackson Laboratory, stock number: 015850) or Tg(OPN1LW-cre)4Yzl/J (Jackson Laboratory, stock number: 0032911) transgenic mice to yield progeny with the genotype Mettl14loxP/+; Rho/HRGP-Cre. Then, the Mettl14loxP/+; Rho/HRGP-Cre mice were crossed with Mettl14flox/flox animals to generate Mettl14flox/flox; Rho -Cre (RKO) or Mettl14flox/flox; HRGP -Cre (HKO) mice. Mettl14flox/flox littermates were used as controls. In addition, to monitor the expression pattern of the Cre enzyme, a tdTomato reporter gene was used (strain name: Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J; Jackson Laboratory, stock number: 007914). In the presence of the Cre enzyme, the stop codon before the tdTomato expression cassette was removed, which permits the expression of tdTomato (red fluorescence) in Cre-positive cells.
Mouse genomic DNA samples were extracted from mouse tails and genotyped using PCR. The floxed Mettl14 alleles and Rho-Cre or HRGP-Cre were genotyped using the corresponding primers (Additional file 4: Table S2). The tdTomato mice were genotyped using primers provided by the JAX mouse service (Additional file 4: Table S2). All amplification reactions were performed using a master mix (Invitrogen, CA, USA) according to the manufacturer’s instructions. The first cycle of PCR consisted of holding at 95 °C for 5 min, followed by 32 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The PCR products were separated by DNA electrophoresis on a 3% agarose gel.
For electroretinographic evaluation of mutants, following overnight dark adaptation, mice were anesthetized with an intraperitoneal injection of xylazine (80 mg/kg) and ketamine (16 mg/kg) in normal saline. Additional anesthetic was given if akinesia was inadequate. The equipment and protocol used here have been described previously . Briefly, the responses of dark-adapted, rod-mediated ERGs to short-wavelength flashes generated by a photopic stimulator over 4.0-log units to the maximum intensity were recorded. Cone-mediated ERGs were recorded in response to white flashes after 10 min of complete light adaptation. The signals were sampled at 0.8 ms intervals and averaged.
For hematoxylin and eosin staining (H&E), enucleated eyes from control and mutant mice were fixed overnight in 1.22% glutaraldehyde and 0.8% paraformaldehyde in 0.08 M phosphate buffer, embedded in paraffin and then cut into 5 μm sections. To ensure that sections used for quantification came from the same eccentricity, the globe was embedded in the same orientation. Sections that encompassed the optic nerve (ON) were selected for staining with H&E according to a standard protocol.
For immunohistochemistry, eyes were removed from euthanized mice by intraperitoneal injection of pentobarbital (75 mg/kg) and by cervical dislocation and fixed in 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 1 h at 4 °C, followed by cryoprotection in 30% sucrose for 2 h. The lenses were removed, and the eyes were embedded in optimal cutting temperature (OCT) solution and sectioned at a 10-μm thickness. After blocking and permeabilization with 10% normal donkey serum and 0.2% Triton X-100 in phosphate buffer for 1 h, the sections were labeled with the primary antibody overnight at 4 °C. The primary antibodies used are shown in Additional file 4: Table S3. The sections were rinsed in PBS three times, Alexa Fluor 594/488-conjugated goat anti-mouse/rabbit secondary antibody (Cat# A11005 and A11008, Invitrogen, Waltham, MA, USA, 1:500 dilution) was applied, and nuclei were counterstained with DAPI (Cat# D8417, Sigma, St Louis, MO, USA) for 1 h at room temperature. Images were captured on a Zeiss LSM 800 confocal scanning microscope. In addition, the fluorescence intensity was quantified using ZEN 2.3 imaging software.
Quantification of cone survival
Retinal whole mounts were prepared as previously described . Briefly, retinas were dissected from enucleated eyes and flattened. The retinas were immersed in 4% PFA for 24 h at 4 °C, blocked in PBS containing 1% bovine serum albumin and 0.5% Triton X-100 and incubated with a polyclonal rabbit anti-L/M-Opsin antibody for 12 h at 4 °C. After several washes, the retinas were incubated with Alexa Fluor 594-conjugated peanut agglutinin (PNA) and an AlexaFluor 488-conjugated goat anti-rabbit secondary antibody (Cat# A11008, Invitrogen, Waltham, MA, USA, 1:250 dilution) for 4 h at room temperature and subsequently washed with PBS. To precisely evaluate the degeneration of cones, the dorsal–ventral orientation of each retina was determined using previously described guidelines , and the retina was then subdivided into 2 sectors with radii of 1 mm and 2 mm. Cones were counted manually in the dorsal squares in each sector to determine the average cone density (cones/mm2). Details about the antibodies and immunohistochemistry procedure are provided in Additional file 4: Table S3.
Apoptotic cell death was detected by the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay according to the manufacturer’s protocol (Cat#11,684,795,910, Roche Diagnostics, Indianapolis, IN, USA) using prepared frozen sections. Images were captured on a Zeiss LSM 800 confocal scanning microscope.
Transmission electron microscopy (TEM)
Anesthetized mice were fixed by transcardial perfusion with PBS followed by 2.5% glutaraldehyde in cacodylate buffer. The eyes were dissected and postfixed in 2.5% glutaraldehyde in cacodylate buffer, pH 7.2, overnight at 4 °C, and then sectioned at 100-mm intervals using a vibratome. The retinal sections were incubated in 1% osmium tetroxide for 1 h, washed in 0.1 M phosphate buffer, dehydrated via an ascending series of ethanol and propylene oxide, and embedded in Epon (25 g of Epon 812, 13 g of dodecenyl succinic anhydride, 12 g of methyl nadic anhydride, and 1 ml of 2,4,6-tris (dimethylaminomethyl) phenol (DMP-30), Electron Microscopy Sciences). Ultrathin Sects. (70 nm) were cut and stained with uranyl acetate and lead citrate. The sections were imaged under a Philips CM120 scanning transmission electron microscope.
RNA m6A dot blot assays
mRNA was purified from total RNA using PolyATtract® mRNA Isolation System III (Cat#Z5300, Promega, Madison, WI, USA) following the protocols supplied with the kit. The mRNAs were subjected to doubling dilution with DEPC water and spotted onto a NC membrane (Cat# HATF00010, Millipore, Billerica, MA, USA). The membranes were dried and then UV crosslinked at 1200 W, blocked with 5% skim milk and incubated with an m6A antibody and with anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (1:5000; Bio-Rad, Hercules, CA, USA), and the signal was developed using SuperSignal West Pico Chemiluminescent Substrate according to the manufacturer’s instructions (Pierce, Rockford, IL, USA). The same RNAs were spotted on the membrane and stained with 0.02% methylene blue in 0.3 M sodium acetate as the loading control.
Methylated RNA immunoprecipitation (MeRIP)
Total RNA was extracted using TRIzol (Cat# Solarbio, Beijing, China). RNA was purified with an EasyPure RNA Purification kit (Cat# ER701-01, TransGen, Beijing, China). The MeRIP assay was carried out with a Magna MeRIP m6A kit (Cat# 17–10,499, Millipore, Billerica, MA, USA) in accordance with the manufacturer’s protocols. m6A modification of particular genes was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with specific primers (primer sequences are shown in Additional file 4: Table S2).
RNA-seq and m6A MeRIP-seq
RNA sequencing analysis was performed on four independent biological replicates from control and RKO retinas at 3 months of age. After harvesting, both retinas for each animal were collected and immediately frozen. The samples were sent to Novogene Corporation Inc. (Beijing, China) for high-throughput MeRIP-seq and RNA-seq on an Illumina HiSeq 2500 platform, and 150 bp paired-end reads were generated. All gene expression values from RNA-seq or m6A MeRIP-seq were changed to a log2 value and further analyzed. Then, a p value of less than or equal to 0.05 was considered to indicate significance.
RNA extraction and RT-qPCR
Retinal total RNA was extracted using TRIzol reagent (Cat# T9424, Sigma, Saint Louis, MO, USA) as recommended by the manufacturer. First-strand cDNA was synthesized using an iScript cDNA Synthesis Kit (Cat# 170–8890, Bio-Rad, Hercules, CA, USA). Quantitative PCR was carried out using iTaq SYBRMix (Cat# 1,725,120, Bio-Rad, Hercules, CA, USA) and a CFX384 Touch Real-Time PCR Detection System (Cat# BJ005303, Bio-Rad, Hercules, CA, USA). Primers were designed using Primer3Plus or obtained from published resources. Additional file 4: Table S2 shows the specific primer sequences used. All target genes were normalized to actin mRNA levels, and the fold change was calculated by performing delta-delta Ct analysis.
Tissues were lysed in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail (Cat# 11,697,498,001, Roche, Redwood City, CA, USA) and a phosphatase inhibitor (Cat# 4,906,845,001, Roche, Redwood City, CA, USA). The protein concentration of the lysates was determined using a DC Protein Assay according to the manufacturer’s instructions (Cat# 500–0122, Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on SDS polyacrylamide gels and transferred onto NC membranes (Cat# HATF00010, Millipore, Billerica, MA, USA). The blots were blocked with 8% nonfat dry milk in Tris-buffered saline (TBS) solution with Tween® 20 detergent for 2 h at room temperature. Then, the membranes were incubated with the primary antibodies in blocking solution overnight at 4 °C. The primary antibodies used for western blotting are shown in Additional file 4: Table S3. The primary antibodies were detected with anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (1:5000; Bio-Rad, Hercules, CA, USA), and the signal was developed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Cat# 34,577, Thermo, Waltham, USA). The relative intensity of the immunoreactive bands was quantified using the gel analysis tool provided in ImageJ software. The intensity of the proteins of interest was normalized to that of GAPDH. At least three independent western blots were conducted, and one typical blot is presented.
Statistical analysis was performed using the GraphPad Prism 6 software. The data sets were tested for a normal distribution using the Shapiro–Wilk test. For normally distributed data, statistical significance was determined by Student’s t-test or ANOVA. If the data were not normally distributed, a nonparametric statistic was used. p-values were calculated with Student’s t-test, one-way or two-way ANOVA followed by Tukey’s, Dunnett’s, or Sidak’s multiple comparisons test as appropriate. p < 0.05 was considered to indicate statistical significance.
Availability of data and materials
The data that supports the findings of this study are available in the supplementary material of this article. Supporting data is included in the Additional files 1, 2, 3, 4. Raw data for m6a sequencing study of Mettl14 RKO retinas was deposited into SRA (Sequence ReadArchive). The access link is as below: https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA756938.
Outer nuclear layer
Inner nuclear layer
Ganglion cell layer
Inherited retinal dystrophy
Congenital stationary night blindness
Rho-Cre mediated knockout
HRGP-Cre mediated knockout
Methylated RNA immunoprecipitation
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labelling
Transmission electron microscopy
Hematoxylin and eosin
Optimal cutting temperature solution
Reverse transcription-quantitative polymerase chain reaction
Integrative Genomics Viewer
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The authors are grateful to the staff of the central laboratory of Sichuan Provincial People’s Hospital and the Lilai Biomedical Experiment Center provided microscopy support. The authors also wish to thank Dr. Huabing Li for Mettl14 floxed allele.
This study was supported by the National Natural Science Foundation of China (82121003, 81970841 to Xianjun Zhu; 82101160 to Yeming Yang), the Department of Science and Technology of Sichuan Province (2021YFS0386, 2021YFS0369, 2021JDGD0036, 2020JDZH0026, 2021JDZH0022), the Department of Science and Technology of Qinghai Province (2022-HZ-814), the Department of Chengdu Science and Technology (2021-YF05-01316-SN), and the CAMS Innovation Fund for Medical Sciences (2019–12 M-5–032), and the China Postdoctoral Science Foundation (2021 M700698). The funders had no role in the study design, data collection and analysis, or preparation of the manuscript.
Ethics approval and consent to participate
All animal experiment protocols were approved by the Institutional Animal Care and Use Committee of Sichuan Provincial People’s Hospital (Chengdu, Sichuan, China) and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
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The authors declare that they have no competing interests.
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Generation of the Mettl14 rod knockout mouse model. Fig. S2. RKO mice showed normal photopic ERG response at 5-month-old. Fig. S3. 2-month-old RKO mice presented normal retinal structure. Fig. S4. Retinal ONL was almost disappeared in aged RKO mice. Fig. S5. Heterozygous RKO mice showed retinal degeneration at 9-month-old. Fig. S6. Inflammatory response and apoptosis in RKO retinas. Fig. S7. Cre-mediated excision of METTL14 in HKO mice. Fig. S8. Visual function analysis of HKO mice.
Enriched GO terms in terms of biological processes.
Enriched GO terms in terms of cellular component.
Information of selected differentially methylated RNA sites. Tables S2. Primers used in this study. Tables S3. Immunological antibodies used in this study.
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Yang, Y., Shuai, P., Li, X. et al. Mettl14-mediated m6A modification is essential for visual function and retinal photoreceptor survival. BMC Biol 20, 140 (2022). https://doi.org/10.1186/s12915-022-01335-x
- Photoreceptor degeneration