Marked for Success: How RNA m⁶A Methylation Fine-Tunes Gut Epithelial Function

Abstract

Post-transcriptional gene regulation—particularly through RNA modifications—plays an essential but understudied role in development, homeostasis, and regeneration of rapidly changing tissues like the mammalian intestinal epithelium. RNA modifications such as N6-methyladenosine (m⁶A) represent a burgeoning area of research in posttranscriptional regulation, with m⁶A being the most abundant modification found in approximately 25% of all mRNA transcripts. Multiple groups have begun to report m⁶A and associated regulation of mRNA fate as critical to key process in the intestinal epithelium. In this review, we synthesize key findings to date into the following 3 categories: m⁶A changes in response to the homeostatic luminal environment, m⁶A as a mediator of stemness in the crypt, and m⁶A as a tool for reacting to inflammation and injury. Over the course of this review, we will demonstrate how m⁶A is uniquely positioned to regulate homeostasis and disease states in the challenging and dynamic environment of the intestinal epithelium.

Summary.

This review synthesizes current knowledge on RNA m⁶A methylation in the intestinal epithelium, examining how writer, reader, and eraser proteins regulate gut homeostasis, regeneration, and disease. It highlights gaps in the field and proposes m⁶A machinery as a potential therapeutic target to explore in inflammatory and neoplastic intestinal conditions.

Most existing research into mechanisms of intestinal epithelial homeostasis and regeneration has focused on the transcriptional, DNA-level changes associated with these processes.^1,2 However, post-transcriptional gene regulation, or gene regulation at the level of RNA, is essential for development, homeostasis, and regeneration in a wide range of tissues.³ Post-transcriptional gene regulation is hypothesized to be especially important in highly dynamic systems where cells undergo rapid changes in identity and function, such as the mammalian intestinal epithelium. Recent proteomics studies underscore this point, demonstrating that RNA and protein abundance are often anti-correlated in rapidly changing tissues such as the intestine.⁴ RNA-protein discordance suggests a prominent role for post-transcriptional regulation of RNA in dynamic tissues. Further investigation into post-transcriptional gene regulation of the intestinal epithelium is essential to define novel mechanisms and therapeutic targets that have not been revealed through the more conventional study of transcriptional biology in the intestine.

One burgeoning area of research in post-transcriptional gene regulation has been RNA modifications, especially the adenosine modification, N6-methyladenosine. Of note, additional forms of RNA methylation such as N1-methyladenosine (m1A) and 5-methylcytidine (m5C) impact for cell fate⁵ and have been implicated in colorectal cancer⁶; however, these mechanisms have yet to be explored in the homeostatic gastrointestinal epithelium. We refer the reader to a comprehensive overview of N6-methyladenosine biology by Murakami et al,⁷ but a brief summary follows. N6-methyladenosine, abbreviated as m⁶A, is the most common covalent modification of RNA. It is found in approximately 25% of all mRNA transcripts and is also deposited on noncoding ribosomal RNA, small nuclear RNA, transfer RNA (tRNA), and chromatin-associated RNAs. The roles of m⁶A on mRNA are the most heavily studied and best understood to date. On mRNA, m⁶A is deposited co-transcriptionally by a large, multi-protein complex comprised of the m⁶A writer proteins.⁸ At the core of this complex are the catalytically active methyltransferase, METTL3, and its catalytically inactive but essential binding partner, METTL14.^9,10 After writing is complete, m⁶A is thought to primarily function by recruiting specific RNA binding proteins that direct downstream transcript fate. The developing convention suggests that m⁶A primarily marks RNA for degradation.^11,12 However, m⁶A also has been implicated in increased RNA stability, as well as translation, alternative splicing, and many other facets of post-transcriptional gene regulation.¹³ The RNA binding proteins that preferentially bind and regulated m⁶A-mRNA are referred to as m⁶A readers. Canonical m⁶A readers are the YTH-domain family of RNA binding proteins. Recently, many other RNA-binding proteins have been identified as m⁶A readers, including the IGF2BP and HNRNP families.14, 15, 16, 17 Finally, 2 m⁶A erasers have been identified: FTO and ALKBH5.^18,19 The existence of these erasers suggests dynamic removal and addition of m⁶A post-transcription. However, ongoing debate questions the relative importance of the m⁶A erasers in significantly altering m⁶A levels in most cell types.^7,20, 21, 22 The process of RNA m⁶A methylation is conserved from bacteria to plants, flies, and mammalian species.23, 24, 25 In multicellular organisms, whole-organism deletion of key components of the methyltransferase complex yield embryonic lethality, underscoring the indispensable role of m⁶A in gene regulation.^24,26 More specifically, m⁶A has been identified as a critical regulator of cell fate, especially within stem cells, where it impacts proliferation, differentiation, survival, and pluri- or multi-potency.⁷

Given the role of m⁶A in stem cells across a variety of other tissues, multiple groups have begun to report m⁶A as critical to key processes in the intestinal epithelium as summarized below. These functions can be split into 3 broad categories: (1) m⁶A as a reaction to changes in the gut luminal environment; (2) m⁶A as a mediator of stemness in the crypt; and (3) m⁶A as a tool for reacting to gut inflammation and injury. Over the course of this review, we will explore these 3 categories sequentially and demonstrate how m⁶A is uniquely positioned to regulate homeostasis and disease states in the challenging and dynamic environment of the intestinal epithelium.

Environmental Changes Reshape the Intestinal Epithelial m⁶A Methylome

Microbiota, nutrients, and oxygen are a few of the many environmental factors that vary considerably across time and space in the intestinal lumen.27, 28, 29 The intestinal epithelium must mount an appropriate adaptive response as the environment around it rapidly changes. As a writeable and erasable means of gene regulation on RNA, m⁶A is one medium epithelial cells may leverage to readjust gene expression in an ever-changing environment. For example, the m⁶A methylome of the intestinal epithelium is broadly altered in germ-free mice as compared with controls.^30,31 In a wild-type control mouse, sequencing of RNA fragments pulled down with an m⁶A-specific antibody (m⁶A-seq) indicates there are as many as 37,000 m⁶A methylation sites across the transcriptome of the cecum.³⁰ Of these, roughly 400 m⁶A sites across 300 transcripts are differentially methylated in germ-free mice as compared with controls with conventional microbiota. This is about 4 times the number of differentially methylated peaks found in the liver of germ-free mice, consistent with the notion that the intestinal epithelium—due to its proximity to the microbiota—is uniquely sensitive to changes in microbial make-up of the gut lumen. Label-free proteomics confirm that several dozen differentially methylated transcripts exhibit changes in protein abundance in germ-free mice compared with controls, with a trend for increased protein abundance in response to increased methylation. These differentially expressed genes are enriched for functions in immunity and the antimicrobial response as well as lipid, vitamin, carbohydrate, and amino acid metabolism. Intriguingly, a subset of these transcripts have altered m⁶A methylation in response to the reintroduction of single bacterial species to germ-free mice, suggesting some transcripts are subject to more direct control of m⁶A methylation by specific bacteria. It remains unclear how the microbiota directly influences RNA methylation. Germ-free mice do not exhibit differential expression of m⁶A writers and erasers in the cecum, where the described experiments were conducted. The m⁶A readers, a much more diverse group of proteins than m⁶A writers, may impart much of the specificity of m⁶A regulation. However, reader expression has not been examined in the context of germ-free mice. Additional experimentation should seek to define the missing links between methylation machinery and microbiota.

Recent data generated outside of the field of intestinal biology challenges the concept that changes in environmental conditions lead to changes in levels of m⁶A methylation on individual transcripts. It is likely that the methylation levels of most m⁶A sites are hard-coded into the cis mRNA sequence rather than being influenced by external factors.^32,33 If this is true, different microbial conditions may still influence the expression of m⁶A-modifided mRNA, but through mechanisms other than altering the stoichiometry of m⁶A methylation levels on individual transcripts. For example, luminal microbes could alter the expression of the m⁶A readers, which determine the fate of methylated transcripts. Nevertheless, studies described in the mouse gut thus far clearly suggest that m⁶A modifies key transcripts in intestinal epithelial cells, and many of these transcripts are uniquely sensitive to changes in luminal microbiota.

In addition to the microbiota, the nutrient content of the gut is in constant flux and the intestinal epithelium must respond appropriately to manage these changes. Given that m⁶A is responsive to luminal conditions and impacts rapid changes in post-transcriptional gene regulation, it is reasonable to hypothesize that the intestinal epithelium leverages m⁶A methylation to react to changes in the nutrient content of the gut lumen. Consistent with this hypothesis, gavaging mice with gliadin plus cholera toxin (a common adjuvant used to trigger intestinal inflammation) leads to a global, 2-fold increase in m⁶A methylation in the duodenum compared with cholera toxin alone.³⁴ Gliadin is a component of gluten and key trigger of celiac disease. Intriguingly, gliadin-mediated increases in m⁶A can directly impact celiac disease phenotypes. A celiac-associated single nucleotide polymorphism (SNP) in the gene exportin 1 (XPO1) is located in an m⁶A methylation site in the XPO1 5′ UTR. Compared with wild-type XPO1, the celiac disease-associated XPO1 variant allele is preferentially methylated in response to gluten exposure and has enhanced translational efficiency. In the presence of gluten, intestinal epithelial cells harboring this SNP increase expression of the celiac disease-associated XPO1 variant, which in turn upregulates epithelial inflammation via nuclear factor κB (NF-κB) and interleukin (IL)-8. Perhaps most striking is the fact that small intestinal biopsies from human patients with celiac disease have increased METTL3 expression and increased global m⁶A, both of which decrease when gluten is removed from the diet of those patients. These data strongly suggest that the m⁶A methylome in the intestinal epithelium is responsive to the nutrient contents of the gut, with substantial implications for human disease. It remains unclear whether the increased methylation in response to gliadin is a direct response to gliadin or an indirect response where gliadin triggers epithelial inflammation, and inflammation then produces m⁶A upregulation. Future studies should parse the relative contributions of gluten vs inflammation to global m⁶A levels in the epithelium and explore the contributions of other dietary changes such as alterations in macronutrients or disease-associated food additives.

These studies collectively demonstrate that changes in the microbial and nutrient content of the gut impact the patterns of m⁶A methylation and the fate of m⁶A-modified mRNA in the intestinal epithelium. They provide enticing evidence that m⁶A enhances the critical ability of the intestinal epithelium to sense and respond to changes in the luminal environment. However, these studies do not explore the fundamental functions of the essential m⁶A machinery in the intestinal epithelium. Which cell types depend on m⁶A most for their function and how does deletion of m⁶A writers, readers, and erasers impact the homeostatic functions or disease states of the intestinal epithelium? Subsequent studies have begun to answer these questions.

m⁶A Writers METTL3 and METTL14 Control Intestinal Stem Cell Survival and Homeostasis

The most direct method of determining the broader role of m⁶A in a tissue is tissue-specific deletion of the m⁶A writers METTL3 or METTL14. These 2 writer proteins are considered co-dependent and essential components of the methylation machinery.^9,10 They have historically been deleted interchangeable to study m⁶A across a wide variety of systems. Although there are conserved roles for METTL3 and METTL14 in maintaining stemness across tissues, METTL3 and METTL14 deletion in any one cell type can yield a wide range of phenotypes, from cell death to enhanced proliferation.35, 36, 37, 38 Therefore, the outcome of deletion of these proteins in a new cell type can never be predicted and must be determined experimentally. A spate of recent publications has detailed ablation of m⁶A in the intestinal epithelium by deleting either METTL3 or METTL14 using the pan-epithelial Villin-Cre or Cre under the control of a variety of stem cell-specific promoters. Each of these studies unequivocally demonstrated that m⁶A is essential for intestinal epithelial homeostasis. However, these papers differ substantially in mechanisms they describe and regions of intestine that are affected. The following section will summarize these papers and highlight key missing data and discrepancies in findings.

Pan-intestinal epithelial m⁶A depletion was first achieved via METTL14 deletion under control of the Villin-Cre promoter, which is active in the mouse intestinal epithelium from embryonic day 12.^39,40 Intestinal epithelial-specific METTL14 knockout (KO) leads to extensive crypt loss and inflammation in the colon, with about one-quarter of pups not surviving to adulthood. Simultaneous METTL14 deletion in homeostatic and facultative stem cells with Lgr5-Cre and Clu-Cre constructs can recapitulate the Villin-Cre phenotype.³⁹ These findings suggest that METTL14 deletion in stem cells alone can explain severe intestinal epithelial defects seen after pan-intestinal epithelial METTL14 deletion. Although the gross phenotype of intestinal epithelial METTL14 KO is consistent across publications, the ascribed mechanism differs. In one, it is proposed that inhibitory NF-κB protein, Nfkbia, is methylated by METTL14 and its subsequent upregulation promotes intestinal epithelial cell death.⁴⁰ In the other, METTL14 methylates and therefore stabilizes gasdermin C transcripts Gsdmc2/3/4, which code for proteins that maintain mitochondrial membrane stability and inhibit apoptosis.³⁹ Although these models are not mutually exclusive, pharmacologic NF-κB inhibition could only minimally restore reduced intestinal organoid formation after METTL14 KO,⁴⁰ whereas ectopic overexpression of GSDMC completely rescued mortality and epithelial cell death after METTL14 KO.³⁹ These data strongly favor the role of METTL14 in inhibiting Gasdermin C (GSDMC) over NF-κB regulation. However, potential issues also arise from this conclusion. Given that METTL14 targets thousands of transcripts, it seems unlikely that the only significant role for this RNA modification in the colonic epithelium is to prevent cytochrome c release from the mitochondria. A dramatic decline in Gsdmc transcript levels is more likely to be a symptom of dying epithelial cells rather than its cause. These data leave questions regarding how m⁶A regulates intestinal stem cell function outside of the apoptotic pathway. Specific mechanisms notwithstanding, multiple studies show that METTL14 is required for colonic stem cell survival, and METTL14 deletion induces an apoptotic pathway dependent on the mitochondrial factor GSDMC. METTL14 likely regulates processes outside of cell death, but these functions remain unknown. Finally, one of the most striking findings from these publications went without explanation. METTL14 deletion caused dramatic cell death in the colonic epithelium but completely spared the small intestine. How could this highly conserved and essential gene regulatory process only be important in the colon? Subsequent studies of METTL3 begin to address this question.

Rather than employ METTL14 deletion as in previous publications, 2 recent publications deleted METTL3 in the intestinal epithelium of adult mice using both the constitutive Villin-Cre and the inducible Villin-CreERT2.^41,42 In contrast to METTL14 deletion, intestinal-epithelial-specific METTL3 deletion in mice produces severe small intestinal defects and organismal death with relative colonic sparing. After inducing METTL3 deletion in adulthood, crypt proliferation dramatically declines, crypts and villi collapse, and mice rapidly lose weight and die within 2 weeks. Two mechanisms for this process of METTL3 KO-mediated death have been described. In one model, METTL3 methylates key pro-stem transcription factors such as Ascl2, maintaining the stemness of LGR5+ stem cells.⁴² In another, METTL3 methylates growth factor pathway transcripts such as Kras, promoting survival of the highly proliferative stem progenitors (aka transit amplifying cells) that maintain epithelial renewal at homeostasis.⁴¹ Neither group was able to rescue the death of METTL3 KO intestinal epithelium with overexpression of pro-stem transcription factors or pro-progenitor growth factors, suggesting that these pathways represent only one part of the essential functions of METTL3 in the small intestinal epithelium. This result is not surprising given that METTL3 targets thousands of transcripts for methylation, and thus the identification of any individual critical pathway downstream of METTL3 is highly unlikely. This challenge can be addressed, in part, by focusing on individual readers described below, which have a narrower range of targets.

In summary, recent publications describing METTL14 and METTL3 KO in the intestinal epithelium demonstrate that METTL14 is essential for colonic epithelial homeostasis and METTL3 is essential for small intestinal epithelial homeostasis. In both cases, METTL3/METTL14 is required for stem cell and/or stem progenitor survival, but the complete mechanism is not yet defined. Furthermore, none of these groups begin to address why pan-intestinal epithelial METTL3 KO and METTL14 KO affect different sections of lower intestine when their deletion is considered equivalent in other tissues. These gaps in knowledge are significant opportunities for additional investigation that should be explored. The intestinal epithelium may be the ideal model for identifying independent functions for the METTL3 and METTL14 writer proteins that have heretofore been considered functionally equivalent.

Readers and an Eraser of m⁶A Regulate Intestinal Regeneration

Intestinal epithelial repair requires a quick response to environmental insult to survive injury. After the initiating injury, epithelial cells must reprogram into a more de-differentiated, fetal-like state to repopulate lost tissue.⁴³ RNA modifications such as m⁶A may be uniquely positioned to assist in the process of intestinal epithelial repair due to their ability to induce rapid changes in expression of genes involved in cell survival and plasticity. However, the m⁶A writers are essential for intestinal homeostasis, making it challenging to leverage METTL3 or METTL14 deletion to study the roles of m⁶A in disease states of the intestinal epithelium. The m⁶A readers and erasers are less conserved across species, have more restricted patterns of expression, and target fewer transcripts than the m⁶A writers.⁷ Due to these characteristics, the readers and erasers are less often essential to tissue survival and have more limited functions, making them much better suited to the study of m⁶A in the context of intestinal epithelial disease.

Deletion of the m⁶A reader YTHDF1 in the intestinal epithelium of mice leads to no significant changes in homeostasis.⁴⁴ However, after radiation-induced injury, YTHDF1-KO epithelium exhibit fewer proliferative epithelial cells and reduced expression of Wnt target genes. Polysome profiling in a YTHDF1-silenced colorectal cancer cell line reveals reduced translation of the transcription factor and Wnt effector, TCF7L2. Overexpression of TCF7L2 rescues growth defects in YTHDF1 KO intestinal enteroids, underscoring the role of m⁶A in supporting intestinal stemness.⁴⁴

IGF2BP1 (IMP1) is another m⁶A reader that has been repeatedly implicated in intestinal epithelial regeneration. IMP1 is a fetal RNA binding protein that is essential for gut development but minimally expressed in adult tissues.⁴⁵ Crypts upregulate Imp1 dramatically after radiation-induced injury, and IMP1 in turn downregulates the epithelial repair process, perhaps to prevent over-proliferation and microadenoma formation as is seen with Wnt-antagonists.^45,46 IMP1 deletion using the HopxCreERT2 driver, which marks regenerative intestinal stem cells after injury, enhances recovery from colonic injury induced by radiation. These mice lose less weight, develop more regenerative crypts, and show higher rates of cell division after injury.⁴⁵ Pan-intestinal epithelial deletion of IMP1 during development with Villin-Cre had similar protective effects in the case of dextran sodium sulfate-induced colitis, possibly through suppressing autophagic flux.^46,47 Interestingly, inducible Villin-CreERT2-mediated deletion of IMP1 in adult mice may instead oppose regeneration by harming intestinal epithelial barrier integrity.⁴⁸ Although multiple publications have defined the specific affinity of IMP1 for m⁶A-modified mRNA,^14,15 the m⁶A-modified targets of IMP1 in the intestinal epithelium have not yet been explored.

HuR is another protein closely associated with IMP1 and recently identified as a potential m⁶A reader.^15,49 HuR stabilizes mRNA, enhancing its expression.⁵⁰ In the homeostatic intestinal epithelium, HuR has been shown to support Wnt signaling by stabilizing transcripts such as the Wnt coreceptor LDL receptor-related protein 6 (LRP6), regulate Paneth cell function by modulating the subcellular localization of Toll-like receptor 2 (TLR2), and promote epithelial repair after injury by stabilizing Caveolin-1 (Cav-1) and Cdc42.51, 52, 53, 54 Like IMP1, the m⁶A-modified targets of HuR in the intestinal epithelium have never been explored.

Finally, the critical role of readers in intestinal epithelial regeneration extends to Drosophila as well. In young Drosophila, the m⁶A reader YT521-B impairs the proliferation of intestinal stem cells during injury, but not at homeostasis, possibly by inhibition of the EGFR-MAPK signaling pathway.⁵⁵ The m⁶A readers comprise a diverse group of proteins regulating the fate of m⁶A-modified mRNA. Many new readers continue to be identified and described, and the majority have yet to be examined in the gut. Each reader represents an opportunity to explore the role of m⁶A in intestinal epithelial homeostasis and is likely to yield phenotypes that provide novel insights into how the intestinal epithelium functions.

In addition to the m⁶A readers, eraser proteins might also promote regeneration in the intestinal epithelium. As with YTHDF1 and IMP1 deletion, intestinal epithelial-specific deletion of the m⁶A eraser, FTO, has no observed effect on gut homeostasis⁵⁶; however, FTO deletion exacerbates colitis. FTO demethylates and stabilizes Cers6 mRNA, which encodes ceramide synthetase. Ceramide synthetase reduces sphingosine-1-phosphate, a compound responsible for recruiting and activating inflammatory macrophages and Th17 T-cells. In this way, it is proposed that FTO reduces inflammatory cell activity during colitis. Intriguingly, decreased FTO expression is associated with increased disease severity in patients with ulcerative colitis. Vedolizumab, a treatment for ulcerative colitis that inhibits immune cell migration into the intestinal mucosa, is more effective in patients with ulcerative colitis with low FTO expression. These data highlight how the study of m⁶A modification in the intestinal epithelium can yield important clinical insights in intestinal disease.

Conclusion

Initial forays into the study of m⁶A and the intestinal epithelium have already yielded significant findings, including a SNP that regulates gluten sensitivity in celiac disease, a novel pathway of programmed cell death in the intestine, and a gene that predicts treatment response in inflammatory bowel disease. Notably, these discoveries come from only 5 years of recently published research (Table 1). This nascent field of studying RNA modifications in the gut still has much to offer investigators looking for new approaches to the study of intestinal epithelial biology. The following is a summary of what is now known in the field and essential gaps and opportunities that remain.

Table 1.

Summary of Key Publications Described in This Review, Divided Into 3 Central Themes

Author, y	Key finding
Environmental changes reshape the intestinal epithelial m6A methylome
Jabs et al, 2020	∼300 transcripts differentially methylated in germ-free mice vs controls, individual bacterial species alter m6A of specific subsets
Olazagoitia-Garmendia et al, 2022	m6A levels increase globally in response to gluten; celiac-associated XPO1 mutant is more methylated and more pro-inflammatory
M6A writers METTL3 and METTL14 control intestinal stem cell survival and homeostasis
Zhang et al, 2022	METTL14 prevents colonic epithelial cell apoptosis by stabilizing inhibitory NF-κB protein, Nfkbia
Du et al, 2022	METTL14 inhibits colonic stem cell apoptosis by stabilizing Gsdmc transcripts, which code for proteins that maintain mitochondrial membrane stability
Liu et al, 2023	METTL3 maintains small intestinal stem cell survival by stabilizing pro-stem transcription factors
Danan et al, 2023	METTL3 is essential for transit amplifying cell survival by promoting translation of growth factor-associated proteins
Readers and an eraser of m6A regulate intestinal regeneration
Han et al, 2020	YTHD1 promotes Wnt-driven stemness after injury via translation of TCF7L2
Chatterji et al, 2018	IMP1 impairs regenerative foci formation after radiation in Hopx-Cre+ facultative stem cells
Chatterji et al, 2019	IMP1 impairs regeneration after colitis, possibly via autophagy inhibition
Parham et al, 2024	IMP1 impairs facultative stemness by suppressing autophagy gene MAP1LC3B
Singh et al, 2020	IMP1 promotes intestinal epithelial barrier integrity after colitis
Zhang et al, 2023	YT521-B impairs proliferation of intestinal stem cells after injury, possibly by inhibiting EGFR-MAPK signaling
Ma et al, 2024	FTO reduces inflammation during colitis by stabilizing Cers6, reducing recruitment of inflammatory immune cells

NOTE. All findings were described in intestinal epithelium.

GSDMC, Gasdermin C; m⁶A, N6-methyladenosine; NF- κB, nuclear factor κB; XPO1, exportin 1.

The studies described in this review allow us to generate a broad understanding of how m⁶A functions in the intestinal epithelium to integrate signals from the luminal environment, maintain intestinal epithelial survival, and regulate inflammation and regeneration (Figure 1).^30,34,39, 40, 41, 42^,44,46, 47, 48^,56 These findings represent a solid foundation, but enormous knowledge gaps persist. Countless writers, readers, and erasers have not been examined in the gut, and their functional effects at a range of timepoints in the healthy and repairing intestine remain unknown.

Figure 1.

Summary of current understanding of role of m⁶A in the intestinal epithelium.Step 1: METTL3 and METTL14 add m⁶A to nascent mRNA, influenced by gut signals like gut microbiota and luminal nutrients. Step 2a: METTL3/14 KO experiments suggest that global m⁶A methylation supports intestinal stem and progenitor cell survival at homeostasis. The readers mediating the essential roles of METTL3/14 remain unidentified; alternatively, METTL3/14 may act through undiscovered reader-independent mechanisms. Step 2b: After injury, m⁶A readers IGF2BP1 and YTHDF1 can promote or inhibit regeneration depending on the reader and context. Step 2c: FTO promotes repair by demethylating and stabilizing select anti-inflammatory transcripts.

Regarding the study of environmental influences on m⁶A, previously employed m⁶A-seq protocols are relatively sensitive for detecting m⁶A sites but lack precise quantitative capabilities for identifying differential methylation.⁷ New techniques that much more accurately determine m⁶A site stoichiometry should be employed to determine the degree to which methylation differs in the intestinal epithelium across different microbial environments.^33,57,58 Furthermore, changes in luminal contents may impact m⁶A dynamics through changes in reader expression rather than changes in m⁶A levels, because m⁶A methylation levels at most sites are likely coded into the RNA sequence itself and thus resistant to environmental changes.^32,33 Future studies should therefore examine changes in reader expression and function in response to environmental changes. These studies should also be expanded to assess the role of luminal contents other than the microbiota. For example, multiple food additives have been implicated in the growing incidence of inflammatory bowel disease.^59,60 Determining if and how these food additives impact gene regulation in the gut via influencing m⁶A might enhance our understanding of the environmental threats that can precipitate intestinal inflammation.

Studies of METTL3 and METTL14 deletion in the gut demonstrate that the former but not the latter is required for small intestinal homeostasis. METTL3 and METTL14 are interdependent members of the same complex and their deletion should have identical effects on m⁶A deposition globally.^9,10 Therefore, it is very surprising that pan-intestinal epithelial METTL3 KO causes severe small intestinal dysfunction, whereas METTL14 KO produces no phenotype. A few studies in other tissue types suggest that METTL3 and METTL14 have roles outside of methylating mRNA. They may regulate the chromatin directly, assist in RNA translation in the cytoplasm, or methylate enhancer- and promoter-associated RNAs.61, 62, 63, 64 These putative m⁶A- or mRNA-independent roles for METTL3 and METTL14 perhaps offer a better explanation for the disparities in small intestine phenotypes upon METTL3 or METTL14 deletion. Perhaps METTL3 deletion leads to a strong small intestinal phenotype when METTL14 deletion does not because METTL3 has some essential function in the small intestine independent of RNA methylation. Published data suggest METTL3 does not maintain intestinal epithelial cell survival through a non-catalytic role,⁴¹ but other possible mechanisms have not been examined. The intestinal epithelium may be an ideal platform for identifying independent functions for METTL3 and METTL14, which would support a paradigm shift in our understanding of how these critical proteins function.

The METTL3/METTL14 KO paradox in the small intestine raises an important question regarding our understanding of m⁶A in the gut, and the study of m⁶A more broadly. Are any of the functions that we assign to m⁶A machinery in the gut—or in any other tissue—truly dependent on m⁶A methylation itself? The answer is more likely yes in the case of XPO1 and celiac disease, where careful experimentation has established a clear potential molecular mechanism. In celiac disease, a SNP in a single m⁶A site increases XPO1 methylation levels and XPO1 translation in association with an increased downstream inflammatory phenotype.³⁴ However, in the case of METTL14 and GsdmC, there is only a correlational connection between m⁶A and GsdmC fate.³⁹ METTL14 deletion eliminates GsdmC RNA methylation and reduces GsdmC expression, but are the outcomes of METTL14 deletion directly dependent on m⁶A? Causational evidence is missing. Such causational evidence might include data showing that genetic ablation of one or more GsdmC methylation sites re-capitulates the phenotype of METTL14 deletion. Additionally, identifying the m⁶A-reader that binds GsdmC in an m⁶A-dependent manner (eg, with cross-linking and immunoprecipitation [CLIP]) would further bolster the mechanistic relationship between METTL14 and its putative target transcript. Although a variety of m⁶A-related proteins have clear, critical roles in the gut, we must be careful in assigning direct relationships between these proteins and m⁶A without data suggesting a precise molecular mechanism. Future research on m⁶A in the gut should emphasize delineating these molecular mechanisms rather than presenting purely correlational data.

Finally, how can the study of m⁶A in the intestine enhance therapy for intestinal disease? This nascent field has already identified potential biomarkers for use in the diagnosis and treatment of celiac disease and ulcerative colitis. Furthermore, identifying new pathways underlying intestinal homeostasis and regeneration aid us in addressing the pathophysiology of mechanistically complex conditions such as inflammatory bowel disease. In addition, m⁶A-regulated pathways could be directly targeted in novel gut-directed therapeutics. For example, selective inhibitors of the writer METTL3 and eraser FTO have shown clinical promise in hematopoietic malignancies.^65,66 If small molecules can manipulate the m⁶A machinery to achieve therapeutic effect in other organ systems, perhaps direct targeting of m⁶A-associated writers, readers, and erasers could also address unmet therapeutic needs in gastrointestinal disease.

Exploring new paradigms in intestinal epithelial gene regulation can address knowledge gaps in the field of intestinal biology. The groundwork in the study of m⁶A in the gut has been established, and early work in this field has been highly rewarding. The next step will be leveraging this groundwork to identify wholly new pathways that are essential to intestinal stem and stem progenitor function. In this way, we can achieve better understanding of the gut and reduce the burden of intestinal disease.