RNA Epigenetics

Methylating RNA

The nucleotide building blocks of RNA contain pyrimidine or purine rings, and each position of these rings can be chemically altered by the addition of various chemical groups. Most frequently, a methyl (–CH3) group is tacked on to the outside of the ring. Other chemical additions such as acetyl, isopentenyl, and threonylcarbamoyl are also found added to RNA bases.

Among the 140 modified RNA nucleotide variants identified, methylation of adenosine at the N6 position (m6A) is the most prevalent epigenetic mark in eukaryotic mRNA. Identified in bacterial rRNAs and tRNAs as early as the 1950s, this type of methylation was subsequently found in other RNA molecules, including mRNA, in animal and plant cells as well. In 1984, researchers identified a site that was specifically methylated—the 3′ untranslated region (UTR) of bovine prolactin mRNA.¹ As more sites of m⁶A modification were identified, a consistent pattern emerged: the methylated A is preceded by A or G and followed by C (A/G—methylated A—C).

The alphabet of RNA consists of at least 140 alternative nucleotide forms.

Although the identification of m6A in RNA is 40 years old, until recently researchers lacked efficient molecular mapping and quantification methods to fully understand the functional implications of the modification. In 2012, we (D.D. and G.R.) combined the power of next-generation sequencing (NGS) with traditional antibody-mediated capture techniques to perform high-resolution transcriptome-wide mapping of m⁶A, an approach we termed m6A-seq.² Briefly, the transcriptome is randomly fragmented and an anti-m⁶A antibody is used to fish out the methylated RNA fragments; the m⁶A-containing fragments are then sequenced and aligned to the genome, thus allowing us to locate the positions of methylation marks.

Analyzing the human transcriptome in this way, we identified more than 12,000 methylated sites in mRNA molecules derived from approximately 7,000 protein-coding genes. The transcripts of most expressed genes, in a variety of cell types, were shown to be methylated, indicating that m6A modifications are widespread. In addition, about 250 noncoding RNA sequences—including well-characterized long noncoding RNAs (lncRNAs), such as the XIST transcripts that have a key role in X-chromosome inactivation—are decorated by m⁶A. In almost all cases, the epigenetic mark was found on adenosines embedded in the predicted A/G—methylated A—C sequence. We found that this pattern was consistently preceded by an additional purine (A or G) and followed by a uracil (U), extending the known consensus sequence to A/G—A/G—methylated A—C—U.²

At the macro level, we found that m6A methylation sites were enriched at two distinct landmarks. The highest relative representation of m6A was found in the stop codon–3′ UTR segment of the RNA, with nearly a third of such methylation found in this sequence just beyond a gene’s coding region. Within the coding regions of the RNA molecules, m6A enrichment mapped to unusually long internal exons; 87 percent of the exonic methylation peaks were found in exons longer than 400 nucleotides. (The average human exon is only 145 nucleotides in length). This pattern of decoration of transcribed RNA suggests that m6A is involved in the mediation of splicing of long-exon transcripts. RNAs transcribed from single­-isoform genes were found to be relatively undermethylated, while transcripts that are known to have multiple isoforms, determined by alternative splicing patterns, were hypermethylated.² Moreover, specific alternative splicing types, such as intron retention, exon skipping, and alternative first or last exon usage, were highly correlated with m⁶A decoration. And silencing the m6A methylating protein METTL3 affected global gene expression and alternative splicing patterns in both human and mouse cells.²

These findings clearly indicate the importance of m6A decoration in regulating the expression of diverse transcripts. Moreover, our parallel study of the human and mouse methylome by m⁶A-seq has uncovered a remarkable degree of conservation in both consensus sequence and areas of enrichment, further supporting the importance of m6A function.² But research into understanding how m⁶A marks themselves are regulated, and how this affects various cellular processes, is only just beginning.

Writers, erasers, and readers

The accumulating findings regarding the cellular consequences of m6A transcriptome decoration led to the search for the mediators that enable m6A to exert its influence. Epigenetic marks are introduced by enzymes and cofactors known as “writers,” and m6A is no exception. This mark is added to RNA by a large protein complex that includes three well-characterized components: METTL3, METTL14, and WTAP.3,4 (See illustration on opposite page.)

The transcripts of most expressed genes, in a variety of cell types, were shown to be methylated.

The reverse process of RNA demethylation is performed by “erasers.” In 2011, one of us (C.H.) and an international group of colleagues identified the first m6A eraser: the fat mass and obesity–associated protein (FTO).5 Four years earlier, three independent studies had discovered that a single-nucleotide polymorphism in the first intron of Fto was strongly associated with body mass index and obesity risk, and studies of mouse models where Fto was deleted or overexpressed further demonstrated its link with altered body weight. The research from the C.H. group showed that silencing the Fto gene or protein increased total m⁶A levels, while overexpression decreased levels of the epigenetic mark.⁵ C.H.’s group later discovered that another protein from the same protein family as FTO, ALKBH5, behaves as an active m⁶A demethylase.⁶ In contrast to the ubiquitous expression of Fto in all tissues, the highest expression level of Alkbh5 was demonstrated in mouse testes. Indeed, Alkbh5-null male mice exhibit aberrant spermatogenesis, probably a result of m⁶A-mediated altered expression of spermatogenesis-related genes.⁶

RNA METHYLATION DYNAMICS: At least 140 alternative RNA nucleotide forms exist. On mRNA, the most common is the methylation of adenosine on the N6 position (m6A). This epigenetic mark is laid down by a “writer” protein complex that includes three well-characterized components: METTL3, METTL14, and WTAP. The reverse process of RNA demethylation is performed by “erasers,” such as the enzymes FTO and ALKBH5.
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These writers and erasers facilitate the dynamic nature of m⁶A methylation, which was shown when we (D.D. and G.R.) demonstrated changes in response to environmental stimuli, such as UV irradiation, heat shock, and exposure to interferon gamma or hepatocyte growth factor.² Once RNA epigenetic modifications are laid down, they are recognized by specific “reader” proteins that bind to the modified nucleotide and mediate enhancement or inhibition of gene expression. In 2012, the G.R. group used methylated and nonmethylated versions of synthetic RNA baits that include the m⁶A consensus sequence to identify such readers of m⁶A.² By preferential binding to the methylated bait, we isolated several specific m6A-binding proteins, including members of the RNA-binding YTH domain family, whose function was previously unknown.²

The finding of the first m6A-binding reader proteins has accelerated the deciphering of the various molecular and cellular processes mediated by m6A marking. In 2014, for example, we (C.H. and colleagues) showed that the human YTH domain family 2 (YTHDF2) reader protein selectively recognizes m⁶A and mediates mRNA degradation.⁷ We identified more than 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs. Binding of YTHDF2 to m⁶A in mRNA results in the translocation of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies in the cytoplasm where mRNA turnover is regulated.

Recently, C.H. and colleagues identified another m6A reader protein, YTHDF1, with a very different function—stimulating protein synthesis by ramping up the efficiency of translation machinery.⁸ The dueling functions of YTHDF2 and YTHDF1 provide a mechanism by which cells can adjust gene expression promptly and precisely to environmental stimuli. Finally, G.R. and his group have identified an additional reader protein, the RNA-binding protein heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1),² which directly binds a set of m⁶A decorated transcripts and mediates alternative splicing.⁹

Clearly, m6A plays diverse roles in regulating cellular function, starting with basic processes such as gene expression, translation, and alternative splicing. As work on this epigenetic mark continues, we will undoubtedly link m6A to numerous phenotypes, and its dysregulation may undergird various diseases and syndromes.

RNA epigenetics in action

Understanding the molecular mechanisms by which m6A regulation controls RNA stability, translation efficiency, and alternative splicing is helping researchers decipher the importance of this new epigenetic mark in physiological and pathological processes. For example, researchers recently showed that translation increases in stressed mice thanks to m⁶A decoration. In 2015, two studies from Cornell University and Weill Cornell Medical College found increased m6A methylation of specific 5′ UTR adenosines in newly transcribed mRNAs as a result of stress-induced nuclear localization of the m⁶A YTHDF2 reader. The researchers suggested that the nuclear YTHDF2 preserves the unique 5′ UTR m⁶A methylation of stress-induced transcripts by limiting the demethylation activity of the FTO eraser. Increased 5′ UTR m⁶A methylation in turn promotes translation of specific transcripts, such as those for the heat shock protein Hsp70. While conventional mRNA translation starts by binding of the ribosome components to a region of the 5′ UTR marked by the unusual nucleotide 7meG (the “cap”), under stress conditions initiation of translation can start farther downstream.¹⁰

DECIDING CELL FATE: Among its many roles in the cell, m6A methylation helps regulate the expression of RNA transcripts that mediate the transition from pluripotency to differentiation. The presence of m6A appears to decrease the stability of transcripts important for maintaining pluripotency, priming the cells for future differentiation. The loss of METTL3, an m6A methlyase component, in mouse embryonic stem cells leads to the cells’ inability to exit the pluripotent state, a lethal outcome in the early embryos.
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In a second study, Weill Cornell Medical College’s Samie Jaffrey, who collaborated on the previous study, led a team that showed m⁶A-methylated mRNAs can be translated in a cap-independent manner. The researchers showed that a specific 5′ UTR m⁶A binds the eukaryotic initiation factor 3 (eIF3), which recruits the ribosomal 43S complex and initiates cap-independent translation. This study also demonstrated increased m⁶A levels in the Hsp70 mRNA that enhanced its cap-independent translation following heat-shock stress.¹¹

Other work has hinted at m⁶A’s role in the regulation of circadian rhythms. Researchers identified m6A sites on many transcripts of genes involved in the regulation of daily cycles. Inhibition of m⁶A methylation by silencing of the METTL3 writer led to circadian period elongation, with altered distribution and processing of the transcripts of the clock genes Per2 and Arntl.¹²

It’s quickly becoming clear that m⁶A decoration has diverse cellular and physiological functions. But perhaps the best illustration of its critical ability to precisely control processes at the cellular level is its involvement in early embryogenesis. Cell-fate decisions are coordinated by alterations in global gene expression, which are orchestrated by epigenetic regulation. Well-established epigenetic marks, such as DNA methylation and histone modifications, are known to mediate embryonic stem cell (ESC) cell-fate decisions, and it turns out that m⁶A modification is no different.

Dynamic m6A RNA markings, the new kid on the epigenetic block, herald the era of tripartite epigenetics where modifications of DNA, RNA, and proteins join hands to fine-tune gene expression and to execute prompt and precise responses to environmental stimuli and stresses.

We (G.R. and collaborators) and other groups recently demonstrated that the m⁶A writer METTL3 is also an essential regulator for termination of mouse embryonic stem cell pluripotency. Knocking out Mettl3 in preimplantation murine epiblasts and in undifferentiated ESCs led to depletion of m6A in mRNAs. Cell viability was not affected, suggesting that m⁶A decoration is not essential for the maintenance of the ESC naive state, but m⁶A marks were critical for early differentiation. The loss of this modification led to aberrant and restricted lineage priming at the post-implantation stage, resulting in early embryonic lethality.¹³ The presence of m⁶A also decreased mRNA stability, including in those transcripts important for maintaining pluripotency. These findings demonstrated, for the first time, an essential function for an mRNA modification in vivo.¹⁴

Beyond mRNA

While m⁶A methylation is most prevalent on mRNAs, this mark also decorates other RNA species. It is well established, for example, that m⁶A is abundant on rRNAs, tRNAs, and small nuclear RNAs (snRNAs), which mediate splicing and other RNA processing and protein synthesis reactions.

More recently, researchers found that the reader protein HNRNPA2B1 binds to m6A marks in a subset of primary microRNA (miRNA) transcripts, recruiting the miRNA-microprocessor complex and promoting primary miRNA processing that is essential for mature miRNA biogenesis.⁹ Not only is the biogenesis of miRNA regulated by m⁶A marking and recruitment of HNRNPA2B1, miRNAs themselves appear to play a role in the placement of the m⁶A epigenetic marks. MiRNAs regulate m⁶A modification in specific transcript sites using a sequence-pairing mechanism where the “seed” sequence of a specific miRNA binds a complementary target sequence in the 3′ UTR of mRNA and directs methylation.¹⁵ The interaction is bidirectional: manipulation of miRNA sequence or expression affects m6A modification also by reducing binding of the METTL3 writer to the target mRNA sites.

Similarly, m6A appears to be involved in structural alterations of mRNAs and lncRNAs to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant RNA-binding protein responsible for mRNA processing. This novel mechanism, termed m⁶A-switch, was shown to affect alternative splicing and abundance of multiple target mRNAs.¹⁶ Taken together, these results demonstrate that m⁶A is an important mark on diverse RNA species.

Dynamic m⁶A RNA markings, the new kid on the epigenetic block, herald the era of tripartite epigenetics where modifications of DNA, RNA, and proteins join hands to fine-tune gene expression and to execute prompt and precise responses to environmental stimuli and stresses. Indeed, m⁶A is just one of 140 modified RNA nucleotides that likely affect the function of the nucleic acid messenger and key cellular actor in diverse ways. Molecular approaches are paving the way for the study of additional RNA modifications.

As the list of RNA epigenetic marks continues to expand, researchers will gain a clearer picture of how diverse cellular processes are regulated. The extremely large repertoire of such modifications is expected to reveal various RNA marks analogous to the known DNA and histone epigenetic marks, and the various modifications of DNA, RNA, and proteins can enrich the language that allows the development, adaptation, and diversity of complex organisms. 

Dan Dominissini is a postdoctoral fellow in Chuan He’s group at the University of Chicago. Gidi Rechavi is a pediatric hematologist-oncologist and a researcher in genetics and genomics at the Chaim Sheba Medical Center in Tel Hashomer, Israel, and a Professor of Hematology at the Sackler School of Medicine at Tel Aviv University. Sharon Moshitch-Moshkovitz, a senior researcher in RNA biology at the Chaim Sheba Medical Center, also contributed to this article.

References

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