Metabolic Regulation of the Epitranscriptome - ACS Chemical Biology

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Metabolic Regulation of the Epitranscriptome Justin M. Thomas, Pedro J. Batista, and Jordan L. Meier ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00951 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 20, 2019

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Metabolic Regulation of the Epitranscriptome

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Justin M. Thomas[a], Pedro J. Batista*[b] and Jordan L. Meier*[a]

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[a]Chemical

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[b]Laboratory

Biology Laboratory, National Cancer Institute, Frederick MD, 21702, USA.. of Cell Biology, National Cancer Institute, Bethesda MD, 20892, USA..

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*Email: [email protected], [email protected]

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Keywords

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Oncometabolite: Metabolites whose abnormal accumulation causes altered signaling that can

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lead to cellular transformation and malignancy.

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Epitranscriptome: Shorthand used to refer to RNA modifications, by analogy with epigenome.

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Cofactor competition: Mechanism of metabolic regulation whereby an endogenous metabolite

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competes with substrate for enzyme active site occupancy, similar to a synthetic inhibitor.

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Cofactor depletion: Mechanism of metabolic regulation whereby depletion of a critical enzyme

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cofactor (e.g. SAM) reduces the activity of enzymes with high Michaelis constant (Km) for that

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cofactor.

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Metabolic regulation of writer localization: Mechanism of metabolic regulation whereby

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cofactor concentrations may affect affinity and interaction of epitransciptomic writers with RNA

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substrates.


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Abstract:

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An emergent theme in cancer biology is that dysregulated energy metabolism may directly

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influence oncogenic gene expression. This is due to the fact that many enzymes involved in gene

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regulation use utilize cofactors derived from primary metabolism, including acetyl-CoA, S-

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adenosylmethionine, and 2-ketoglutarate. While this phenomenon was first studied through the

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prism of histone and DNA modifications (the epigenome), recent work indicates metabolism can

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also impact gene regulation by disrupting the balance of RNA post-transcriptional modifications

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(the epitranscriptome). Here we review recent studies that explore how the metabolic regulation

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of writers and erasers of the epitranscriptome (FTO, TET2, NAT10, MTO1, METTL16) helps

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shape gene expression through three distinct mechanisms: cofactor inhibition, cofactor depletion,

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and writer localization. Our brief survey underscores similarities and differences between the

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metabolic regulation of the epigenome and epitranscriptome, and highlights fertile ground for

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future investigation.



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Introduction

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The ability of metabolism to influence gene regulatory mechanisms in human cells is an

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increasingly well-established phenomenon. While canonical metabolic sensors such as growth

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factor receptors and nutrient-sensitive kinases have been known for decades, a more recent

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finding is that metabolites may play a direct role in transcriptional regulation by influencing

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chromatin structure in the nucleus. Early studies in this area demonstrated that disruption of

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acetyl-CoA metabolism can inhibit histone acetylation, implying that under certain conditions

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acetyl-CoA becomes rate-limiting for histone acetyltransferases.1-2 These findings were

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remarkable, as they suggested the enzymatic that add (“writers”) and remove (“erasers”) of

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histone modifications (many of which use primary metabolites as cofactors) may serve as a link

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between environmental cues and powerful epigenetic mechanisms of gene regulation in

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eukaryotes. This concept of ‘metabolic regulation of epigenetics,’ as well examples of its

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relevance to physiology and disease, has been summarized excellently elsewhere.3-5

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Five years ago in ACS Chemical Biology, we reviewed three mechanisms by which

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metabolites had been found to directly impact the activity of enzymes involved in epigenetic

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signaling: 1) competitive inhibition, 2) cofactor depletion, and 3) localized enzymatic effects.6

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Intriguingly, these metabolic mechanisms are not necessarily limited to enzymes involved in DNA

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and histone modifications but may also encompass other classes of cofactor-utilizing enzymes

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that play a signaling role in cancer, including the writers and erasers of RNA modifications. The

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ability of RNA to function as a direct sensor of metabolic information is well-precedented in

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bacteria, where riboswitches drive nutrient-dependent gene expression programs.7 Related is the

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fact that over 100 modified nucleobases exist in human coding and non-coding RNA and, just like

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histone modifications, many of these modifications are derived from primary metabolic cofactors.

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Similarly, just as histone modifications are associated with changes in gene transcription, RNA

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modifications can alter gene expression via diverse mechanisms.8-10 The importance of these

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mechanisms is highlighted by the critical roles described for RNA modification in both


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development and in disease.11 In this perspective we summarize recent efforts to understand the

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metabolic regulation of RNA modifications, also known as the epitranscriptome. Rather than

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providing a comprehensive review of the metabolic regulation of RNA modifications, this article

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aims to focus attention on a few contemporary examples of studies of metabolically-sensitive

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writer and eraser enzymes as case studies for defining this phenomenon. In addition to reviewing

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these critical studies, our brief survey highlights similarities and differences between the metabolic

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regulation of the epigenome and epitranscriptome, and suggests future areas for further

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exploration.

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Metabolic Regulation of the Epitranscriptome by Competitive Inhibition (FTO, TET2,

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NAT10)

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The most disease-relevant mechanism by which metabolism has been shown to directly influence

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protein and nucleic acid modifications is via the production of competitive metabolites. The

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hallmark example of this was found in 2008, when large-scale glioblastoma and acute myeloid

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leukemia genome sequencing efforts identified driver mutations in the active-sites of the primary

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metabolic enzymes isocitrate dehydrogenase 1 and 2 (IDH1/IDH2).12-14 Mechanistic studies

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revealed these genetic lesions cause formation of heterodimeric IDH enzymes imbued with novel

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ability to transform 2-ketoglutarate into (R)-2-hydroxyglutarate (2-HG).15 (R)-2-HG accumulates

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to millimolar levels in tumors and patient tissues, allowing it to competitively inhibit Fe(II)--

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ketoglutarate-dependent dioxygenase enzymes involved in control of histone demethylation, and

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DNA hydroxymethylation.16-17 The ability to promote malignant transformation by influencing gene

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regulatory mechanisms has led (R)-2-HG to be termed an “oncometabolite.”

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In contrast to their ability to trigger cancer initiation, an increasingly appreciated

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paradoxical property of oncometabolites is their ability to exert deleterious effects on mature tumor

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progression. This is exemplified by the finding that IDH mutant glioblastomas are associated with

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a longer median overall survival than other variants of the disease,18-19 and that high levels of (R)-



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2-HG can inhibit cell growth and confer collateral inhibitor vulnerabilities across a number of cell

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types.20-22 However, it is relatively unknown how specific dioxygenases communicate information

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about (R)-2-HG levels to gene expression programs involved in regulation of cell growth. Su and

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coworkers examined the effects of (R)-2-HG on the enzyme FTO, an Fe(II)--ketoglutarate

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dioxygenase which catalyzes the removal of N6-methyladenosine (m6A) and N6,2’-O-

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dimethyladenosine (m6Am) from RNA.23-25 Careful analysis of gene expression data found that

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sensitivity to (R)-2-HG correlated with high expression of FTO. This led the authors to hypothesize

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these cell lines may be addicted to active FTO-dependent m6A demethylation, which preliminary

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data from an earlier study had suggested.26 Consistent with this hypothesis, treatment of AML cell

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lines with (R)-2-HG led to an increase overall m6A levels. (R)-2-HG also caused an increase in

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the proteolytic and thermal stability of FTO, providing additional support for ligand occupancy and

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a direct oncometabolite-FTO interaction.27-28 Transcriptome-wide mapping of m6A sites found (R)-

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2-HG treatment increased the methylation of 5’-untranslated regions (5’-UTRs) and coding

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sequences of many transcripts, including the master oncogenic transcription factor Myc.

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Expression of a synthetic gene construct incorporating the Myc 5’-UTR was activated by FTO

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overexpression and inhibited by (R)-2-HG treatment. These and additional experiments support

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an overall model in which competitive inhibition of FTO by (R)-2-HG increases levels of m6A in

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the Myc 5’-UTR, accelerating decay of Myc mRNA and limiting pro-growth gene expression

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programs (Figure 1). (R)-2-HG also increased m6A -dependent decay of CEBPA, a transcription

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factor necessary for FTO expression, providing an additional mechanism that amplifies

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oncometabolite-dependent m6A disequilibrium.

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These findings have several implications for the biology of IDH mutant cancers. First, they

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suggest that disruption of the FTO/ m6A/Myc axis may be an important contributor to the improved

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prognosis observed in cancer harboring driver mutations in IDH enzymes. Since this initial study

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focused on examining the response of m6A to (R)-2-HG levels raised by dosing or ectopic

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expression of IDH mutants, it will be important to determine whether m6A equilibrium is similarly


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altered in AML clinical specimens harboring neomorphic IDH mutations. Furthermore, the finding

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that (R)-2-HG antagonizes an m6A demethylation program required for optimal cell growth

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suggests inhibition of FTO may be a viable therapeutic strategy in IDH mutant cancers. FTO has

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recently been found to play an oncogenic role in a variety of mutationally-defined AMLs, and it is

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tempting to speculate that the partial inhibition of FTO caused by (R)-2-HG accumulation may

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render IDH mutant cancers especially sensitive to small molecule FTO inhibitors.29 Related to this

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thought, the finding that after malignant transformation (R)-2-HG production actually limits cell

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growth suggests in some contexts these cancers may be better treated by targeting collateral

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vulnerabilities30 (such as FTO), rather than directly targeting production of (R)-2-HG itself using

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inhibitors of mutant IDH.31-32 Future work will be required to test this hypothesis, as well as to

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assess how m6A levels correlate with clinical response. Another question raised by these studies

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is whether modulation of m6A may play a role in gene regulation in other settings marked by (R)-

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2-HG accumulation. In addition to other IDH mutant cancers,33 elevated (R)-2-HG levels have

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been observed in the absence of IDH mutation, and hpoxia has been found to increase levels of

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(S)-2-HG.34-35 It is likely that examination of m6A and m6Am in these diverse settings will yield

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additional insights into the metabolic regulation of RNA modifications and their role in malignancy.

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Extending our analogy, in the metabolic regulation of epigenetic signaling, cofactor

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competitive metabolites have been found to alter the activity of many different classes of

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chromatin-modifying enzymes.36 Similarly, early evidence suggests these active-site dependent

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mechanisms may also apply to several classes of RNA modifiers. The TET proteins are Fe(II)--

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ketoglutarate-dependent enzymes that oxidize 5-methylcytidine to 5-hydroxymethylcytidine

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(hm5C) in DNA and RNA.37-38 (R)-2-HG has been validated as a competitive inhibitor of TET2,39

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and TET2 and IDH mutations are mutually exclusive in AML,40 suggesting they carry out

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redundant functions. A recent study found that ectopic overexpression of neomorphic IDH1 and

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IDH2 mutants altered the ability of TET2 to catalyze formation of hm5C in RNA.41 Coupled with

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recent evidence that intragenic hm5C is present in mRNA and can functionally alter translation,42



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these findings suggest decreased levels of hm5C in RNA may contribute to gene expression

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changes in IDH mutant AML.

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Projecting beyond 2-HG, many methyltransferases have been found to be sensitive to S-

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adenosylmethionine (SAM)-competitive metabolites such as S-methylthioadenosine (MTA),

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which accumulates in methylthioadenosine phosphorylase (MTAP)-deficient cancers.43 It is

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plausible that elevated levels of MTA may also alter the activity of RNA methyltransferase

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enzymes, which are responsible for >40 eukaryotic RNA modifications that have been postulated

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to be derived from SAM. Recently an acetylated nucleobase, N4-acetylcytidine (ac4C), was found

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to occur in human transfer RNA (tRNA) and ribosomal RNA (rRNA).44-45 Our group applied an

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unbiased chemical proteomic approach46 to discover that NAT10, the enzyme responsible for

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ac4C deposition, binds tightly to the metabolic feedback inhibitor CoA.47 This suggests NAT10

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may be sensitive to the cellular acetyl-CoA/CoA ratio, which is known to be decreased by fasting

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and bioenergetic stress.1-2 Consistent with this hypothesis, one group has reported that disruption

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of acetyl-CoA biosynthesis reduces levels of ac4C in yeast,48 while another has found that

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starvation of C. elegans inhibits ac4C levels in tRNA.49 Although a definitive role for ac4C in gene

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regulation awaits determination, this modification is highly conserved and has been implicated in

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control of tRNA stability, rRNA biogenesis, and mRNA stability.45, 50-51 Intriguingly, the activity of

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NAT10 has also been implicated in premature aging syndromes.52 Given the well-characterized

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links between metabolism, histone acetylation, and aging,53-54 the development of new methods

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to study RNA acetylation55-56 may help determine whether this modification also play a role in the

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gene regulatory response to stimuli such as caloric restriction and Warburg metabolism.

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Metabolic Regulation of the Epitranscriptome by Cofactor Depletion (MTO1, GTPBP3)

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A second way in which metabolism can influence the activity of cofactor-utilizing enzymes is via

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cofactor depletion.6 In this mechanism, depletion of a critical metabolic cofactor (e.g. SAM)

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reduces the activity of enzymes possess a high Michaelis constant (Km) for that cofactor.


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Despite much study, it remains undetermined whether cofactor depletion alone can cause rate-

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limiting decreases in the activity of enzymes that utilize SAM, 2-ketoglutarate, and acetyl-CoA,

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or whether these activity changes are reliant on coincidental increases in competitive

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metabolites such as SAH, 2-HG, and CoA (described above). However, a recent study by

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Morscher and coworkers suggests that cofactor depletion can powerfully repress the activity of

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RNA-modifying enzymes responsible for synthesis of the tRNA modification 5-

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taurinomethyluridine (τm5U).57 These studies were spurred by the curious observation that

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cellular knockout of SHMT2, but not other folate one-carbon (1C) biosynthetic enzymes,

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enhanced extracellular acidification of colon cancer cell grown in culture. SHMT2 plays a critical

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role in folate 1C metabolism by mediating the serine-dependent transformation of THF to

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methylene-THF in the mitochondria.58 Hypothesizing that the observed increase in glycolysis

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could be due to impaired mitochondrial function, Morscher et al. used a variety of functional

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assays to determine that deletion of SHMT2 impairs cellular respiration by decreasing the

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abundance of the complex I, IV, and V subunits of the mitochondrial electron transport chain.

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Careful metabolic detective work and feeding studies further revealed methylene-THF as the

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key metabolite whose depletion by SHMT2 knockout was responsible for respiratory chain

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deficiency. After ruling out mechanisms such as mitochondrial DNA damage, the authors next

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considered the possibility that SHMT2/methylene-THF depletion may specifically inhibit

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translation in the mitochondria.

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Mitochondria contain localized ribosomes and tRNAs that are responsible for the

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translation of 13 proteins in humans, including several enzymes in complex I, IV, and V of the

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electron transport chain.59 To test the hypothesis that methylene-THF may be required for

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protein translation in this organelle, a mitochondria-specific ribosome profiling method was

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developed. The deep coverage and high resolution afforded by this method revealed that

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SHMT2 knockout decreased levels of actively translating ribosomes at mRNAs encoding

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several subunits of the respiratory complex, in part due to a high rate of stalled ribosomes at the



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aminoacyl-tRNA acceptor site of specific lysine and leucine codons (LysAAG and LeuUUG).57 No

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stalling was observed at LysAAA and LeuUUA codons, suggesting that SHMT2 deficiency causes

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cells difficulty reading the 3’ guanosine of certain codons. In humans, decoding of codons

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ending in A/G in split codon boxes is facilitated by modifications of the tRNA anticodon which

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allow non-Watson-Crick base-pairing with the codon 3’ base. Within mitochondria this is

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accomplished by tRNA wobble base modifications derived from 1C metabolism, including 5’-

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taurinomethyluridine (τm5U). To determine whether τm5U may be sensitive to mitochondrial

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methylene-THF levels, the authors assessed this modification by LC-MS and found that τm5U

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and its 2-thio derivative were depleted to undetectable levels in SHMT2 knockout cell lines. This

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reduction was not due to reduced taurine in these cell lines and could be rescued by re-

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expression of wild-type SHMT2 or administration of the methylene-THF precursor sarcosine.

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Isotopic labeling experiments revealed the metabolic source of the τm5U methyl group was

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methylene-THF rather than SAM, denoting τm5U as the first macromolecular modification

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directly derived from folate in mammals. MTO1 is a member of the enzyme complex responsible

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for formation of τm5U in human mitochondrial tRNAs. Consistent with the hypothesis that

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methylene-THF can become rate-limiting for τm5U deposition, knockout of the τm5U biosynthetic

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enzyme MTO1 increased occupancy at the same 3’-guanosine-containing codons affected by

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SHMT2 depletion. In addition, evidence suggests disrupted folate 1C metabolism may inhibit

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τm5U -mediated translation of respiratory chain proteins in several additional contexts, including

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mitochondrial disorders, neonatal folate deficiency, and the therapeutic administration of

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antifolates to treat immune disorders and cancer.57 Valuably, parallel investigations of τm5U in

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mitochondrial disease made similar findings regarding its regulation by folate metabolism and

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dietary taurine, providing additional perspective and validation.60-61

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The discovery of a mitochondrial translational program regulated by 1C metabolism

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raises several questions for the field. First, what is the molecular basis for the sensitivity of the

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τm5U modification to folate metabolism? In this regard it is noteworthy that 5-formylcytidine


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(f5C), a 1C metabolism-derived RNA modification found at the 5’-anticodon of mitochondrial

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tRNAMet, was unaffected by SHMT2 ablation.41 This suggests that metabolic cofactor depletion

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can selectively impact the activity of a subset of RNA-modifying enzymes, a concept similar to

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the spectrum of metabolic sensitivity displayed by chromatin-modifying enzymes such as

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histone acetyltransferases. In the case of τm5U and f5C, this selectivity may stem from a

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different range of cellular metabolite concentrations attained their 1C metabolite cofactors

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(methylene-THF and SAM), differential enzyme kinetic parameters such as cofactor Km, or

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some combination thereof. Further biochemical and metabolomic analyses will likely be

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informative in this regard. An additional question raised by these studies is whether metabolic

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mechanisms regulate codon-specific translation via RNA modifications in other contexts. As the

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role of tRNA modifications in the cellular stress response has been detailed elsewhere,10, 62-63

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we limit ourselves here to one potentially informative example. In yeast, levels of sulfur-

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containing amino acids modulate levels of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), a

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modified nucleobase found at the 5’-anticodon (wobble base) of several cytosolic tRNAs.64 65-67

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While nutrient-sensitive mcm5s2U deposition has not yet been reported in humans, it is known

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that altered levels of the enzymes which synthesize this modification can alter gene expression

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through codon-biased translation in neurodegenerative diseases as well as cancer.65, 68 In the

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future it will be interesting to assess whether mcm5s2U is dysregulated in cancer contexts that

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are highly dependent on cysteine,69-70 or responsive to changes in acetyl-CoA and SAM levels,

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which also contribute to formation of this modification and contain cysteine-derived elements.71

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Complex indirect regulation is also plausible, as exemplified by the recent finding that 1-

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methyladenosine in tRNA regulates translation and is responsive to glucose-dependent gene

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expression changes.72 Beyond these tRNA examples, gene translation may also be altered by

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mRNA modifications, some of which have already established to be metabolically sensitive

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(including m6A detailed above).73 It is likely that as novel roles for RNA modifications in gene



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expression continue to be discovered, additional examples of metabolic regulation of the

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epitranscriptome by cofactor depletion mechanism will emerge.

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Metabolic Regulation of Epitranscriptome Writer Localization. (METTL16)

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Besides globally turning the activity of their writer and eraser enzymes, metabolic stimuli can also

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manifest surprising effects at specific genomic and transcriptomic loci.6 One of the first examples

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of this mechanism was characterized in chromatin, where genomic localization of the SAM

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synthetase MAT2A was found to be required for histone methylation and transcriptional

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repression of specific genes.74 This supported a model in which MAT2A supplies a localized pool

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of SAM to protein methyltransferases to aid transcriptional regulation. Extending this paradigm to

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the epitranscriptome, Pendleton and coworkers recently demonstrated that metabolic

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perturbations can reduce localized RNA methyltransferase activity at the MAT2A transcript level,

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and in so doing elicit a protein-RNA interaction which aids maintenance of cellular SAM levels.75

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MAT2A is a metabolic enzyme responsible for catalyzing the ATP-dependent formation of

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SAM from methionine. Early studies had found that the abundance of the MAT2A mRNA

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increases upon methionine depletion, presumably to allow cells to maintain SAM levels during

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nutrient stress.76 Through a series of elegant experiments, Pendleton et al determined that

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reduced cellular SAM triggers the splicing of an intron-retained MAT2A isoform that is normally

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unstable. Splicing of the intron-retained isoform increases the abundance of the MAT2A mRNA,

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and was further found to depend on a proximal hairpin in the 3’-UTR which is marked by the

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methylated RNA nucleobase m6A. Mutational studies in a model MAT2A construct demonstrated

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that methionine-dependent splicing an m6A site in the proximal hairpin. Additional studies found

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that methylation did not depend on the major mRNA methyltransferase METTL3,77 but rather

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catalyzed by a relatively uncharacterized methyltransferase, METTL16. Unexpectedly, while

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methionine depletion and METTL16 knockdown both decreased m6A in MAT2A’s 3’-UTR, only

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methionine depletion prompted splicing. This led the authors to propose a model where inefficient


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methylation due to low SAM causes METTL16 to linger on the MAT2A transcript. This metabolite-

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dependent increase in occupancy then promotes METTL16-driven splicing through m6A-

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independent effects. Consistent with this, the authors found that overexpression of a METTL16

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catalytic domain mutant, but not an RNA-binding domain mutant, could trigger splicing. Similarly,

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using MS2-tethering to increase METTL16 binding to a synthetic MAT2A construct increased

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splicing. Mutational analyses together with RNA immunoprecipitation were used to determine that

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the METTL16 methyltransferase domain was required for methionine-dependent changes in

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MAT2A mRNA occupancy, while the highly conserved VCR domain was found to be required for

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splicing. A number of additional studies to further characterize the regulation of m6A by METTL16

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led to the discovery that METTL16 can have a broad influence on m6A in messenger RNA through

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a combination of direct and indirect effects, a subset of which may be mediated by MAT2A.75 In

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another example of scientific convergence, analogous findings regarding the feedback regulation

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of MAT2A mRNA stability were also made by Shima and coworkers.78

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The discovery of a functional metabolite-dependent protein-RNA interaction provides

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fodder for future investigation of several areas. First, these studies suggest that the absence of

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an enzyme cofactor can cause RNA modification writers such as METTL16 to functionally act as

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readers, binding to unmodified substrates constitutively, and triggering non-catalytic functions

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such as nucleation of protein-protein interactions. This recalls studies that have suggested

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chromatin-modifying histone deacetylases can also act as functional readers.79 The ability of this

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mechanism to influence activity would also be expected to be highly dependent on kinetic

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mechanism of these enzymes; for example, many acetyltransferases are known exhibit ordered

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binding of acetyl-CoA followed by peptide substrate,80 while many methyltransferases are known

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to interact with substrate first, or show no preference for ordered binding.81 In the future it will be

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interesting to understand the biochemical basis for high occupancy RNA recognition by METTL16,

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as well as whether this paradigm applies to other classes of writers and erasers of nucleic acid

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and protein modifications. Of note, many writers and erasers of modifications are multifunctional



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enzymes with important non-catalytic activities, including the major mRNA methyltransferase

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METTL3 as well as the histone acetyltransferase EP300.82-83 Another question pertains to how

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prevalent the splicing-based stabilization of nuclear intron isoforms is as a homeostatic

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mechanism. A recent report found this mechanism regulates OGT, a glycosyltransferase that

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utilizes the glucose-derived metabolite UDP-O-GlcNAc as a metabolic precursor.84 The ability of

303

cells to rapidly increase OGT levels may explain observations that glucose-derived histone

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acetylation is sensitive nutrient stress but protein O-GlcNAc-ylation is not.2, 85 This intimates a

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larger point, which is the ability of homeostatic mechanisms to cause unexpected effects on

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metabolically-derived protein and nucleic acid modifications. For example, based on the

307

mechanism above MAT2A knockdown would be expected to have a more substantial effect on

308

SAM levels than methionine deprivation. Thus, studying the metabolic regulation of an RNA

309

modification under these two different conditions may lead to dramatically different conclusions.

310

This suggests that care should be taken to directly measure metabolite levels, as well as probe

311

for the existence of homeostatic feedback mechanisms based on writer, reader, eraser, or

312

biosynthetic enzyme abundance in future studies of metabolically-regulated signaling. While

313

homeostatic feedback adds a layer of complexity onto the paradigm of metabolic control, the

314

studies above provide a model for unraveling these mechanisms and also powerfully illustrate the

315

ability of such studies to yield new insights into gene regulation.

316 317

Conclusions

318

Here we have reviewed several recent examples of how metabolic mechanisms can influence the

319

posttranscriptional chemical modification of RNA, also known as the epitranscriptome. In stem

320

cells populations, metabolism is rewired in order to maintain a balance between pluripotency and

321

differentiation, a strategy co-opted by several cancers. Compared to proteins and DNA, RNAs

322

undergoes a larger spectrum of enzyme-catalyzed modifications, many of which are sourced from

323

a more complex pool of metabolites. Exemplifying this, the mitochondrial wobble base


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324

modification τm5U is derived from methylene-THF and taurine, while the cytosolic modification

325

mcm5s2U derives from cysteine, SAM, and acetyl-CoA (Figure 4). This suggests the potential for

326

RNA modifications to have a more complex relationship with metabolism than protein

327

modifications. This attribute is potentially compounded by the sequential establishment of many

328

RNA modifications, affording additional opportunity for interplay. Furthermore, the participation of

329

multiple classes of RNA molecules, all targets of writers and erasers, in the processes leading to

330

protein production further contributes to a complex interaction between the metabolic environment

331

in the cell and posttranscriptional regulation of gene expression. Another difference between

332

epigenetic and epitranscriptomic regulation is the relative time-scale of these processes. While

333

chromatin modifications can be copied during replication and inherited by daughter cells, RNA

334

modifications will be lost upon turnover. The limited lifetime of cellular RNAs may alleviate the

335

necessity for eraser enzymes for many of these modifications, with the consequence that the

336

direct influence of metabolism on RNA modifications may manifest primarily through writer

337

enzymes. Conversely, while changes in the equilibrium of chromatin modifications require

338

extended time periods to be established, the relatively rapid turnover of RNA provides a

339

mechanism for more rapid response to environmental stimuli, as exemplified by the FTO/Myc

340

axis. Defining how these kinetic differences influence the scope of RNA modification-dependent

341

regulatory responses, how metabolism may affect RNA stability and turnover, and whether rapid

342

changes in RNA modifications can help facilitate long-term changes to the epigenome will be

343

important to future understanding of this phenomenon. Finally, it is important to emphasize that

344

we have highlighted only a small number of examples where metabolism has been found to

345

directly influence the writers and erasers of RNA modifications. Additional metabolic mechanisms

346

of epitranscriptomic regulation have been proposed86 and characterized,87 and many more novel

347

examples no doubt await discovery. Future efforts to unravel these mechanisms will benefit from

348

the development and application of new tools to study metabolic regulation. Methods for rapid

349

metabolite induction and depletion will be important to study fast biological responses prior to



350

engagement of pleiotropic effects.88-89 Improved approaches for mapping mRNA modifications

351

may enable their effects on gene regulation to be assessed with greater resolution.90-91 New tools

352

for the high-throughput analysis of metabolite-protein interactions involved in the regulation of

353

writer and eraser enzymes will be useful to identify new pathways susceptible to this

354

phenomenon.92-94 Further efforts in this area have the potential to provide substantial insights into

355

the signaling role of metabolites and the role they play in the powerful gene regulatory

356

mechanisms that underlie fundamental biology, medicine, and human disease.

357 358

Acknowledgements.

359

We sincerely apologize to the many authors whose work could not be included due to space

360

limitations. This work was supported by National Institutes of Health, National Cancer Institute,

361

Center for Cancer Research (ZIABC011766 and ZIABC011488).

362


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363


Figure Legends.

364 365

Figure 1. Metabolic regulation of the epitranscriptome by competitive inhibition. Mutant IDH1

366

produces high levels of (R)-2-hydroxyglutarate which competitively inhibits FTO, a Fe(II)/2-

367

ketoglutarate-dependent dioxygenase that erases m6A in mRNA. Increased m6A levels in mRNAs

368

encoding oncogenes such as Myc reduce transcript stability and impede cell growth.

369 370

Figure 2. Metabolic regulation of the epitranscriptome by cofactor depletion. Inhibition of SHMT2

371

depletes 5,10-methylene-THF, which serves as the 1C donor for the enzymatic formation of 5-

372

taurinomethyluridine (τm5U). This reduces τm5U in a subset of mitochondrial tRNAs and causes

373

decreased translation of electron transport chain proteins.

374 375

Figure 3. Metabolic regulation of epitranscriptome writer localization. Under conditions of nutrient

376

abundance (left), METTL16 catalyzes methylation of an mRNA encoding the SAM biosynthetic

377

enzyme MAT2A, limiting its splicing and reducing MAT2A mRNA stability. When SAM levels are

378

low (right), METTL16 inefficiently methylates the MAT2A mRNA but forms a stable interaction.

379

This triggers splicing and mRNA stabilization that allows more MAT2A to be produced and for

380

cellular SAM homeostasis to be maintained.

381 382

Figure 4. Diverse metabolic sources of RNA modifications. m6A = N6-methyladenosine, mcm5s2U

383

= 5-methoxycarbonylmethyl-2-thiouridine, ac4C = N4-acetylcytidine.

384



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