The metabolic baton: conducting the dance of N6-methyladenosine writing and erasing.

The modification N6-methyladenosine (m6A) plays an important role in determining the functional output of gene expression programs. Throughout the transcriptome, the levels of m6A are tightly regulated by the opposing activities of methyltransferases and demethylases, as well as the interaction of modified transcripts with m6A-dependent RNA-binding proteins that modulate transcript stability, often referred to as writers, erasers, and readers. The enzymatic activities of both writers and erasers are tightly linked to the cellular metabolic environment, as these enzymatic reactions rely on metabolism intermediaries as cofactors. In this review, we highlight the examples of intersection between metabolism and m6A-dependent gene regulation and discuss the different contexts where this interaction plays important roles.

The modification landscape of Pseudomonas aeruginosa tRNAs.

RNA modifications have a substantial impact on tRNA function, with modifications in the anticodon loop contributing to translational fidelity and modifications in the tRNA core impacting structural stability. In bacteria, tRNA modifications are crucial for responding to stress and regulating the expression of virulence factors. Although tRNA modifications are well-characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa, remains limited. Here, we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in Escherichia coli, which enabled us to identify similar modifications in P. aeruginosa Our analysis supports a substantial degree of conservation between the two organisms, while also uncovering potential sites of tRNA modification in P. aeruginosa tRNAs that are not present in E. coli The mutational signature at one of these sites, position 46 of tRNAGln1(UUG) is dependent on the P. aeruginosa homolog of TapT, the enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) modification. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA-modifying enzymes, some of which play roles in determining virulence and pathogenicity.

The thiol methyltransferase activity of TMT1A (METTL7A) is conserved across species.

Although few resistance mechanisms for histone deacetylase inhibitors (HDACis) have been described, we recently demonstrated that TMT1A (formerly METTL7A) and TMT1B (formerly METTL7B) can mediate resistance to HDACis with a thiol as the zinc-binding group by methylating and inactivating the drug. TMT1A and TMT1B are poorly characterized, and their normal physiological role has yet to be determined. As animal model systems are often used to determine the physiological function of proteins, we investigated whether the ability of these methyltransferases to methylate thiol-based HDACis is conserved across different species. We found that TMT1A was conserved across rats, mice, chickens, and zebrafish, displaying 85.7%, 84.8%, 60.7%, and 51.0% amino acid sequence identity, respectively, with human TMT1A. Because TMT1B was not found in the chicken or zebrafish, we focused our studies on the TMT1A homologs. HEK-293 cells were transfected to express mouse, rat, chicken, or zebrafish homologs of TMT1A and all conferred resistance to the thiol-based HDACIs NCH-51, KD-5170, and romidepsin compared to empty vector-transfected cells. Additionally, all homologs blunted the downstream effects of HDACi treatment such as increased p21 expression, increased acetylated histone H3, and cell cycle arrest. Increased levels of dimethylated romidepsin were also found in the culture medium of cells transfected to express any of the TMT1A homologs after a 24 h incubation with romidepsin compared to empty-vector transfected cells. Our results indicate that the ability of TMT1A to methylate molecules is conserved across species. Animal models may therefore be useful in elucidating the role of these enzymes in humans.

The modification landscape of P. aeruginosa tRNAs.

RNA modifications have a substantial impact on tRNA function, with modifications in the anticodon loop contributing to translational fidelity and modifications in the tRNA core impacting structural stability. In bacteria, tRNA modifications are crucial for responding to stress and regulating the expression of virulence factors. Although tRNA modifications are well-characterized in a few model organisms, our knowledge of tRNA modifications in human pathogens, such as Pseudomonas aeruginosa, remains limited. Here we leveraged two orthogonal approaches to build a reference landscape of tRNA modifications in E. coli, which enabled us to identify similar modifications in P. aeruginosa. Our analysis revealed a substantial degree of conservation between the two organisms, while also uncovering potential sites of tRNA modification in P. aeruginosa tRNAs that are not present in E. coli. The mutational signature at one of these sites, position 46 of tRNAGln1(UUG) is dependent on the P. aeruginosa homolog of TapT, the enzyme responsible for the 3-(3-amino-3-carboxypropyl) uridine (acp3U) modification. Identifying which modifications are present on different tRNAs will uncover the pathways impacted by the different tRNA modifying enzymes, some of which play roles in determining virulence and pathogenicity.

Rewiring of RNA methylation by the oncometabolite fumarate in renal cell carcinoma.

Metabolic reprogramming is a hallmark of cancer that facilitates changes in many adaptive biological processes. Mutations in the tricarboxylic acid cycle enzyme fumarate hydratase (FH) lead to fumarate accumulation and cause hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC is a rare, inherited disease characterized by the development of non-cancerous smooth muscle tumors of the uterus and skin, and an increased risk of an aggressive form of kidney cancer. Fumarate has been shown to inhibit 2-oxoglutarate-dependent dioxygenases (2OGDDs) involved in the hydroxylation of HIF1α, as well as in DNA and histone demethylation. However, the link between fumarate accumulation and changes in RNA post-transcriptional modifications has not been defined. Here, we determine the consequences of fumarate accumulation on the activity of different members of the 2OGDD family targeting RNA modifications. By evaluating multiple RNA modifications in patient-derived HLRCC cell lines, we show that mutation of FH selectively affects the levels of N6-methyladenosine (m6A), while the levels of 5-formylcytosine (f5C) in mitochondrial tRNA are unaffected. This supports the hypothesis of a differential impact of fumarate accumulation on distinct RNA demethylases. The observation that metabolites modulate specific subsets of RNA-modifying enzymes offers new insights into the intersection between metabolism and the epitranscriptome.

The Methyltransferases METTL7A and METTL7B Confer Resistance to Thiol-Based Histone Deacetylase Inhibitors.

Histone deacetylase inhibitors (HDACi) are part of a growing class of epigenetic therapies used for the treatment of cancer. Although HDACis are effective in the treatment of T-cell lymphomas, treatment of solid tumors with this class of drugs has not been successful. Overexpression of the multidrug resistance protein P-glycoprotein (P-gp), encoded by ABCB1, is known to confer resistance to the HDACi romidepsin in vitro, yet increased ABCB1 expression has not been associated with resistance in patients, suggesting that other mechanisms of resistance arise in the clinic. To identify alternative mechanisms of resistance to romidepsin, we selected MCF-7 breast cancer cells with romidepsin in the presence of the P-gp inhibitor verapamil to reduce the likelihood of P-gp-mediated resistance. The resulting cell line, MCF-7 DpVp300, does not express P-gp and was found to be selectively resistant to romidepsin but not to other HDACis such as belinostat, panobinostat, or vorinostat. RNA-sequencing analysis revealed upregulation of the mRNA coding for the putative methyltransferase, METTL7A, whose paralog, METTL7B, was previously shown to methylate thiol groups on hydrogen sulfide and captopril. As romidepsin has a thiol as the zinc-binding moiety, we hypothesized that METTL7A could inactivate romidepsin and other thiol-based HDACis via methylation of the thiol group. We demonstrate that expression of METTL7A or METTL7B confers resistance to thiol-based HDACis and that both enzymes are capable of methylating thiol-containing HDACis. We thus propose that METTL7A and METTL7B confer resistance to thiol-based HDACis by methylating and inactivating the zinc-binding thiol.

The thiol methyltransferase activity of TMT1A (METTL7A) is conserved across species.

Although few resistance mechanisms for histone deacetylase inhibitors (HDACis) have been described, we recently demonstrated that TMT1A (formerly METTL7A) and TMT1B (formerly METTL7B) can mediate resistance to HDACis with a thiol as the zinc-binding group by methylating and inactivating the drug. TMT1A and TMT1B are poorly characterized, and their normal physiological role has yet to be determined. As animal model systems are often used to determine the physiological function of proteins, we investigated whether the ability of these methyltransferases to methylate thiol-based HDACis is conserved across different species. We found that TMT1A was conserved across rats, mice, chickens, and zebrafish, displaying 85.7%, 84.8%, 60.7% and 51.0% amino acid sequence identity, respectively, with human TMT1A. Because TMT1B was not found in the chicken or zebrafish, we focused our studies on the TMT1A homologs. HEK-293 cells were transfected to express mouse, rat, chicken, or zebrafish homologs of TMT1A and all conferred resistance to the thiol-based HDACIs NCH-51, KD-5170 and romidepsin compared to empty vector-transfected cells. Additionally, all homologs blunted the downstream effects of HDACi treatment such as increased p21 expression, increased acetylated histone H3, and cell cycle arrest. Increased levels of dimethylated romidepsin were also found in the culture medium of cells transfected to express any of the TMT1A homologs after a 24 h incubation with romidepsin compared to empty-vector transfected cells. Our results indicate that the ability of TMT1A to methylate molecules is conserved across species. Animal models may therefore be useful in elucidating the role of these enzymes in humans.

Cryptic splice mutation in the fumarate hydratase gene in patients with clinical manifestations of Hereditary Leiomyomatosis and Renal Cell Cancer.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an autosomal dominant condition characterized by the development of cutaneous and uterine leiomyomas and risk for development of an aggressive form of papillary renal cell cancer. HLRCC is caused by germline inactivating pathogenic variants in the fumarate hydratase (FH) gene, which encodes the enzyme that catalyzes the interconversion of fumarate and L-malate. We utilized enzyme and protein mobility assays to evaluate the FH enzyme in a cohort of patients who showed clinical manifestations of HLRCC but were negative for known pathogenic FH gene variants. FH enzyme activity and protein levels were decreased by 50% or greater in three family members, despite normal FH mRNA expression levels as measured by quantitative PCR. Direct Nanopore RNA sequencing demonstrated 57 base pairs of retained intron sequence between exons 9 and 10 of polyadenylated FH mRNA in these patients, resulting in a truncated FH protein. Genomic sequencing revealed a heterozygous intronic alteration of the FH gene (chr1: 241498239 T/C) resulting in formation of a splice acceptor site near a polypyrimidine tract, and a uterine fibroid obtained from a patient showed loss of heterozygosity at this site. The same intronic FH variant was identified in an unrelated patient who also showed a clinical phenotype of HLRCC. These data demonstrate that careful clinical assessment as well as biochemical characterization of FH enzyme activity, protein expression, direct RNA sequencing, and genomic DNA sequencing of patient-derived cells can identify pathogenic variants outside of the protein coding regions of the FH gene.

The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1.

The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.

Identification of Protein-RNA Interactions in Mouse Testis Tissue Using fRIP.

During development, cells must quickly switch from one cell state to the next to execute precise and timely differentiation. One method to ensure fast transitions in cell states is by controlling gene expression at the post-transcriptional level through action of RNA-binding proteins on mRNAs. The ability to accurately identify the RNA targets of RNA-binding proteins at specific stages is key to understanding the functional role of RNA-binding proteins during development. Here we describe an adapted formaldehyde RNA immunoprecipitation (fRIP) protocol to identify the in vivo RNA targets of a cytoplasmic RNA-binding protein, YTHDC2, from testis, during the first wave of spermatogenesis, at the stage when germ cells are shutting off the proliferative program and initiating terminal differentiation ( Bailey et al., 2017 ). This protocol enables quick and efficient identification of endogenous RNAs bound to an RNA-binding protein, and facilitates the monitoring of stage-specific changes during development.

Dual Inhibition of Histone Deacetylases and the Mechanistic Target of Rapamycin Promotes Apoptosis in Cell Line Models of Uveal Melanoma.

Purpose: Over 90% of uveal melanomas harbor pathogenic variants of the GNAQ or GNA11 genes that activate survival pathways. As previous studies found that Ras-mutated cell lines were vulnerable to a combination of survival pathway inhibitors and the histone-deacetylase inhibitor romidepsin, we investigated whether this combination would be effective in models of uveal melanoma. Methods: A small-scale screen of inhibitors of bromodomain-containing protein 4 (BRD4; OTX-015), extracellular signal-related kinase (ERK; ulixertinib), mechanistic target of rapamycin (mTOR; AZD-8055), or phosphoinositide 3-kinase (PI3K; GDC-0941) combined with a clinically relevant administration of romidepsin was performed on a panel of uveal melanoma cell lines (92.1, Mel202, MP38, and MP41) and apoptosis was quantified by flow cytometry after 48 hours. RNA sequencing analysis was performed on Mel202 cells treated with romidepsin alone, AZD-8055 alone, or the combination, and protein changes were validated by immunoblot. Results: AZD-8055 with romidepsin was the most effective combination in inducing apoptosis in the cell lines. Increased caspase-3 and PARP cleavage were noted in the cell lines when they were treated with romidepsin and mTOR inhibitors. RNA sequencing analysis of Mel202 cells revealed that apoptosis was the most affected pathway in the romidepsin/AZD-8055-treated cells. Increases in pro-apoptotic BCL2L11 and decreases in anti-apoptotic BIRC5 and BCL2L1 transcripts noted in the sequencing analysis were confirmed at the protein level in Mel202 cells. Conclusions: Our data suggest that romidepsin in combination with mTOR inhibition could be an effective treatment strategy against uveal melanoma due in part to changes in apoptotic proteins.

Small RNAs are modified with N-glycans and displayed on the surface of living cells.

Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These "glycoRNAs" were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.

You Get a Modification, and You Get a Modification Everyone Gets a Modification!

In this issue of Molecular Cell,Sun et al. (2020) identify ERK-mediated phosphorylation of the m6A methyltransferase complex as a regulatory mechanism for m6A and pluripotency and highlight the potential of this interaction as a target for cancer therapy.

RNA: a double-edged sword in genome maintenance.

All organisms must safeguard the integrity of their DNA to avoid deleterious consequences of genome instability, which have been linked to human diseases such as autoimmune disorders, neurodegenerative diseases and cancer. Traditionally, genome maintenance has been viewed largely in terms of DNA-protein interactions. However, emerging evidence points to RNA as a key modulator of genome stability, with seemingly opposing roles in promoting chromosomal instability and protecting genome integrity. Unravelling the mechanistic and contextual basis of this duality will not only improve our understanding of the interfaces between RNA and the genome but will also provide important insights into how disrupted RNA metabolism contributes to disease origin, laying the foundation for targeted intervention.

PIRCh-seq: functional classification of non-coding RNAs associated with distinct histone modifications.

We develop PIRCh-seq, a method which enables a comprehensive survey of chromatin-associated RNAs in a histone modification-specific manner. We identify hundreds of chromatin-associated RNAs in several cell types with substantially less contamination by nascent transcripts. Non-coding RNAs are found enriched on chromatin and are classified into functional groups based on the patterns of their association with specific histone modifications. We find single-stranded RNA bases are more chromatin-associated, and we discover hundreds of allele-specific RNA-chromatin interactions. These results provide a unique resource to globally study the functions of chromatin-associated lncRNAs and elucidate the basic mechanisms of chromatin-RNA interactions.

Metabolic Regulation of the Epitranscriptome.

An emergent theme in cancer biology is that dysregulated energy metabolism may directly influence oncogenic gene expression. This is due to the fact that many enzymes involved in gene regulation use cofactors derived from primary metabolism, including acetyl-CoA,  S-adenosylmethionine, and 2-ketoglutarate. While this phenomenon was first studied through the prism of histone and DNA modifications (the epigenome), recent work indicates metabolism can also impact gene regulation by disrupting the balance of RNA post-transcriptional modifications (the epitranscriptome). Here we review recent studies that explore how metabolic regulation of writers and erasers of the epitranscriptome (FTO, TET2, NAT10, MTO1, and METTL16) helps shape gene expression through three distinct mechanisms: cofactor inhibition, cofactor depletion, and writer localization. Our brief survey underscores similarities and differences between the metabolic regulation of the epigenome and epitranscriptome, and highlights fertile ground for future investigation.

It's complicated m6A-dependent regulation of gene expression in cancer.

Cellular function relies on multiple pathways that are coordinated to ensure the proper execution of gene expression networks. Failure to coordinate the multiple programs active in the cell can have catastrophic consequences and lead to diseases such as cancer. At the post-transcriptional level, RNA modifications play important roles in the regulation of gene expression. N6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification and has gained increasing interest in the last few years as a dynamic regulator of RNA metabolism. Modifications regulate all stages of the RNA life cycle, from transcription to decay. Recent studies have pointed to the role of RNA methylation in cancer initiation and progression, and aberrant modification has served as a biomarker of early-stage diagnosis in several cancers. Here, we review the regulation of m6A, disruptions to methylation-dependent pathways that influence carcinogenesis, and potential avenues for m6A-related therapeutic strategies.