‘(m6)A’ stands for Autoimmunity Reading, Writing, and Erasing RNA modifications during inflammation (2025)

Abstract

Covalent modifications of RNA that regulate gene expression post-transcriptionally, in particular N6-methyladenosine (m6A), are emerging as important regulators of autoimmune responses. This article highlights new findings describing the functional diversity and specificity of m6A modifications and their regulation in the context of autoimmunity.

The m6A pathway and autoimmunity

RNA epigenomic modifications have been recognized for decades, but only recently have they begun to be evaluated in further depth in the context of the immune system [1]. RNA methylation of adenosine at nitrogen-6 (N6-methyladenosine, m6A) is among the most common. This modification is not static, but rather can be dynamically regulated by methyltransferases that catalyze m6A addition (known as writers), demethylases that reverse this process (erasers), and RNA binding proteins (readers) that recognize m6A-modified RNA and influence RNA export, stabilization, translation, among other events. Hence, the myriad consequences of m6A modifications depend on fine-tuned interactions of the m6A machinery with target mRNAs [1].

Early studies of the m6A pathway in immunity were performed in the context of viral infections (Box 1) [2]. While inflammation is required to protect against pathogens, dysregulation of immune homeostasis can result in auto-inflammation. Autoimmune diseases vary in the affected organs and clinical manifestations, and are categorized as organ-specific [e.g., multiple sclerosis (MS)] or systemic [e.g., systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA)]. There is accumulating evidence to suggest that the m6A pathway may promote pathogenesis or progression of certain autoinflammatory disorders [3, 4]. Specifically, expression of certain m6A machinery (ALKBH5, YTHDF2) is decreased in PBMC from SLE and RA patients [3, 4]. These links are at present just observational. Nevertheless, since RNA m6A modifications constitute a significant regulatory layer of prominent immune function, it is highly plausible that dynamic changes in the RNA ‘methylome’ potentially contribute in a meaningful way to certain autoimmune disorders. Here, we discuss how the m6A machinery functions in immune contexts to regulate autoimmune manifestations in pre-clinical settings, and implications for human disease.

Box 1 – m6A machinery and innate immunity.

m6A modifications influence viral pathogenesis by acting directly on viral transcripts and by regulating host mRNAs [2]. For example, m6A modifications were identified in IFNB1, MAVS, TRAF3, TRAF6 among other mRNAs [15, 16]. Whereas m6A deposition decreases the stability of IFNB1 in fibroblasts infected with multiple viruses [e.g., CMV, adenovirus, or vesicular stomatitis virus (VSV)], this modification increases nuclear export and translation of MAVS, TRAF3, TRAF6 in VSV-infected macrophages. m6A modifications can additionally suppress recognition of viral RNA through pattern-recognition receptors expressed on human dendritic cells (DCs) [2]. Recent studies highlighted the role of m6A machinery in other immune cells as well (e.g DCs, natural killer cells) [1, 17].

The m6A machinery in T cell-driven autoimmunity : m6A writers

Mice with a deficiency in the core m6A writer METTL3 in CD4+ T cells (Mettl3fl/flCd4Cre+) were found to exhibit impaired homeostatic proliferation and differentiation of naïve CD4+ T cells in lymph nodes. This was linked to reduced colitis in a T cell transfer model of intestinal inflammation [5]. In these mice, mRNAs encoding SOCS-family JAK-STAT inhibitors (Socs1, Socs3, Cish) were subject to m6A modification, with prolonged RNA half-life and increased SOCS protein expression in Mettl3-deficient T cells [5] (Figure 1A). This led to impairments in IL-7-induced JAK-STAT activation in CD4+ T cells, suppressing proliferation and differentiation into pathogenic effector T cells [5]. While Mettl3fl/flCd4Cre+ mice developed normally, they exhibited intestinal inflammation starting at 3 months of age [6]. A similar phenotype was observed in mice lacking METTL14, the catalytic partner of METTL3 (Mettl14fl/flCd4Cre+) [7]. From these findings, it was suggested that Tregs had reduced suppressive capability as a result of impaired capacity to methylate RNA targets. Indeed, mice lacking Mettl3 in Tregs (Mettl3fl/flFoxp3Cre+) showed a similar increase in the expression of Socs family genes compared to control Mettl3fl/fl mice, with reduced IL-2 signaling and concomitantly reduced suppressive function [6] (Figure 1B). Therefore, Mettl3fl/flFoxp3Cre+ developed systemic autoimmune responses [6] Taken together, these data suggest an important role for RNA m6A modifications in modulating gene expression that alters CD4+ T cell homeostatic proliferation and Treg suppressive functions through the control of JAK-STAT signaling, at least in mice.See AlsoAI Transcription: A Quick Start GuideME - Doctor of Philosophy (PhD) / Master of Philosophy (MPhil)

Figure 1 – Functional diversity and specificity of m6A machinery: implications for autoimmunity.

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It is known that the abundance of immediate-early inducible genes including SOCS gene family is regulated by mRNA degradation. Since many of these transcripts are m6A-modified, m6A modifications may be a means by which immediate-early gene transcripts are targeted for degradation in T cells. In humans, the expression of SOCS genes is reduced in CD4+ T cells from patients with psoriasis compared to healthy donors, with analogous findings in CD4+ T cells from MS patients during relapse compared to remission [8, 9]. Whether m6A-dependent degradation of SOCS genes is functionally responsible for these observations is in need of further assessment.

m6A machinery in T cell-driven autoimmunity : m6A readers and erasers

In general, the m6A writers METTL3/14 are the core enzymes of this pathway, and thus their deletion impacts a multitude of downstream events. m6A readers, by contrast, appear to determine the consequences of m6A deposition in a more context-specific manner. Therefore, linking individual m6A readers to observed phenotypes is important for understanding – and ultimately exploiting –the m6A machinery in the context of autoimmunity. The best-studied m6A readers belong to the 5-member YTH family (YTHDF1/2/3, YTHDC1/2) [1]. In addition to YTH domain-containing proteins, the insulin-like growth factor 2 mRNA-binding family (IGF2BP1/2/3, known as IMPs) have the capacity to recognize m6A-modified RNA [1]. Generally, RNA-binding by YTH readers results in mRNA degradation, whereas IGF2BPs seem to primarily promote the stability of client mRNAs [1]. However, the nature of the specific m6A reader(s) implicated in CD4+ T cell homeostasis and Treg suppressive function described above remains unknown [5, 6].

The discovery that m6A writers operate in CD4+ T cells raised the intriguing possibility that m6A erasers might control certain T cell functions in an analogous manner. The ability to reverse this process could allow for more dynamic regulation of the transcriptome in response to changing conditions. Indeed, deletion of the m6A eraser ALKBH5 in CD4+ T cells (Alkbh5fl/flCd4Cre+) was found to confer protection against autoimmunity as illustrated in models of colitis and experimental autoimmune encephalomyelitis (EAE) [10]. Specifically, Alkbh5-deficient CD4 T cells exhibited increased m6A modification on Ifng and Cxcl2 transcripts, which were associated with reduced mRNA stability and protein levels relative to Alkbh5fl/fl controls [10] (Figure 1A). In EAE, these modifications were linked to attenuated CD4+ T cell responses and decreased neutrophil recruitment into the central nervous system. However, these studies did not report whether ALKBH5 impacted SOCS-family mRNAs (as seen with METTL3/14 deletion), and therefore, this axis will require further investigation.

Whereas METTL3 can regulate Treg function in mice [6], Alkbh5-deficient Tregs (Alkbh5fl/flFoxp3Cre+) did not show evidence for altered suppressive capabilities, as they developed EAE similarly to controls [10]. Thus, other demethylases are likely to participate in regulating Treg functions. One possible candidate is FTO, a ubiquitous and well-characterized demethylase. However, CD4+ T cells express far less Fto than Alkbh5 upon TCR activation [10] and FTO is dispensable for T cell development and EAE pathogenesis in Ftofl/flCd4Cre+ mice [10]. The finding that m6A writers, but not erasers, sustain Treg suppressive activities might imply that this an important homeostatic mechanism to limit autoimmune inflammation. These results also suggest that there is considerable selectivity and nuance in the roles that m6A modifiers play in T cells.

The m6A machinery in B cell-driven autoimmunity

To date, the consequences of m6A modifications on B cell-driven autoimmunity are poorly characterized. METTL3 supports differentiation of T follicular helper cells (TFH), which provide help to B cells in germinal centers (GC) [11]. Indeed, Mettl3fl/flCd4Cre+ mice show decreased m6A modifications which are associated with destabilization of TFH signature genes (e.g., Tcf7) [11]. In addition, METTL3 can regulate GC B cell proliferation directly, described in models where Mettl3 was specifically deleted in GC B cells (AID-Mettl3fl/fl) [12]. The latter exhibited a decrease in expression of oxidative phosphorylation and proliferation-related genes (e.g., Myc) [12]. Like Socs, Myc is an early-inducible gene known to be m6A-modified. In this setting, two distinct m6A readers were responsible for the observed phenotypes; IGF2BP3 (IMP3) regulated Myc expression, whereas YTHDF2 controlled oxidative phosphorylation genes [12]. While progress has been made in understanding the role of RNA modifications in GC maintenance, much is yet to be learned, including whether the m6A pathway regulates B cell-driven autoimmunity. Broadly, since m6A machinery clearly contributes to GC formation, we predict that blocking this pathway may be a viable strategy to alleviate some features of antibody-driven autoimmunity.

The m6A machinery in autoimmunity driven via non-hematopoietic cells

Beyond their well-established role in providing structural support to organs, epithelial and mesenchymal cells are now becoming more appreciated to be actively involved in immune responses, both at the level of the affected tissue as well as within in the LN. Therefore, m6A-driven posttranscriptional changes in structural cells potentially have broad consequences on orchestrating immune responses. Recently, our group found that a non-canonical m6A reader IGF2BP2 (IMP2) functions in mouse fibroblasts and human renal epithelial cells to mediate IL-17 signaling and induction of signature target genes [13]. In particular, Il6 and Lcn2 (Lipocalin-2, aka NGAL) mRNAs were strongly IMP2-dependent; both genes are important drivers of autoimmune inflammation in autoantibody-induced glomerulonephritis (AGN), an IL-17-driven immune setting [14]. Indeed Imp2−/− mice were protected from disease in AGN compared with wildtype mice [13]. IL-17 mediates many of its signals via the CCAAT/enhancer binding proteins (C/EBP) b and d. Unexpectedly we found that mRNAs encoding C/EBPb and C/EBPd are also m6A-modified which was required for IMP2-binding, concomitant transcript stabilization and ultimately C/EBP protein expression within kidney during AGN [13]. Accordingly, IMP2-dependent regulation of Cebpd and Cebpb perpetuates IL-17-driven kidney inflammation (Figure 1C).

Although there are many YTH-family m6A readers, only IMP2 promoted IL-17 signaling [13]. The basis of this specificity is not understood, since all the currently identified m6A readers recognize the same motif DRA*CH (D=G/A/U, R=G/A, H=U/A/C, * indicates methylated A) [1]. RNA secondary structures adjacent to reader binding motifs may dictate the recruitment of specific m6A readers to client RNAs. It is also likely that tissue-specific expression of m6A readers contributes to selectivity of m6A sensing. Therefore, m6A recognition may be regulated by cis-acting sequences in the mRNA itself, and by trans-acting tissue-specific factors. Cumulatively all these factors likely influence the outcome of m6A deposition.

Concluding Remarks

Our understanding of the m6A pathway in immune system regulation is in its infancy. Given the diversity of immune mRNAs that are m6A modified, many more m6A-driven phenotypic changes during autoimmunity are likely to be discovered, some of which might be exploited for clinical benefit. Nevertheless, major knowledge gaps exist regarding the specificity, selectivity, and regulation of m6A machinery. Nevertheless, perhaps it might eventually be possible to link specific m6A modifications to corresponding pathology. But even if not, we might still earn an “(m6) A for effort.”

Acknowledgments

Funding:

NIH supported S.L.G. (R01-AI147383).

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