Splice epitranscriptomics and DNA damage repair together: ALKBH5-m6A-SF3B1 regulation in leukemic transformation.

In this issue, Ciesla et al.1 report a translation regulation through ALKBH5-mediated 5'-UTR m6A demethylation of the SF3B1 transcript during leukemic transformation. The SF3B1 protein maintains efficient splicing and expression of transcripts encoding DNA damage repair components to restrain excessive DNA damage.

A lncRNA from the FTO locus acts as a suppressor of the m6A writer complex and p53 tumor suppression signaling.

N6-methyladenosine (m6A) of mRNAs modulated by the METTL3-METTL14-WTAP-RBM15 methyltransferase complex and m6A demethylases such as FTO play important roles in regulating mRNA stability, splicing, and translation. Here, we demonstrate that FTO-IT1 long noncoding RNA (lncRNA) was upregulated and positively correlated with poor survival of patients with wild-type p53-expressing prostate cancer (PCa). m6A RIP-seq analysis revealed that FTO-IT1 knockout increased mRNA m6A methylation of a subset of p53 transcriptional target genes (e.g., FAS, TP53INP1, and SESN2) and induced PCa cell cycle arrest and apoptosis. We further showed that FTO-IT1 directly binds RBM15 and inhibits RBM15 binding, m6A methylation, and stability of p53 target mRNAs. Therapeutic depletion of FTO-IT1 restored mRNA m6A level and expression of p53 target genes and inhibited PCa growth in mice. Our study identifies FTO-IT1 lncRNA as a bona fide suppressor of the m6A methyltransferase complex and p53 tumor suppression signaling and nominates FTO-IT1 as a potential therapeutic target of cancer.

FMRP phosphorylation modulates neuronal translation through YTHDF1.

RNA-binding proteins (RBPs) control messenger RNA fate in neurons. Here, we report a mechanism that the stimuli-induced neuronal translation is mediated by phosphorylation of a YTHDF1-binding protein FMRP. Mechanistically, YTHDF1 can condense with ribosomal proteins to promote the translation of its mRNA targets. FMRP regulates this process by sequestering YTHDF1 away from the ribosome; upon neuronal stimulation, FMRP becomes phosphorylated and releases YTHDF1 for translation upregulation. We show that a new small molecule inhibitor of YTHDF1 can reverse fragile X syndrome (FXS) developmental defects associated with FMRP deficiency in an organoid model. Our study thus reveals that FMRP and its phosphorylation are important regulators of activity-dependent translation during neuronal development and stimulation and identifies YTHDF1 as a potential therapeutic target for FXS in which developmental defects caused by FMRP depletion could be reversed through YTHDF1 inhibition.

RNA m5C oxidation by TET2 regulates chromatin state and leukaemogenesis.

Mutation of tet methylcytosine dioxygenase 2 (encoded by TET2) drives myeloid malignancy initiation and progression1-3. TET2 deficiency is known to cause a globally opened chromatin state and activation of genes contributing to aberrant haematopoietic stem cell self-renewal4,5. However, the open chromatin observed in TET2-deficient mouse embryonic stem cells, leukaemic cells and haematopoietic stem and progenitor cells5 is inconsistent with the designated role of DNA 5-methylcytosine oxidation of TET2. Here we show that chromatin-associated retrotransposon RNA 5-methylcytosine (m5C) can be recognized by the methyl-CpG-binding-domain protein MBD6, which guides deubiquitination of nearby monoubiquitinated Lys119 of histone H2A (H2AK119ub) to promote an open chromatin state. TET2 oxidizes m5C and antagonizes this MBD6-dependent H2AK119ub deubiquitination. TET2 depletion thereby leads to globally decreased H2AK119ub, more open chromatin and increased transcription in stem cells. TET2-mutant human leukaemia becomes dependent on this gene activation pathway, with MBD6 depletion selectively blocking proliferation of TET2-mutant leukaemic cells and largely reversing the haematopoiesis defects caused by Tet2 loss in mouse models. Together, our findings reveal a chromatin regulation pathway by TET2 through retrotransposon RNA m5C oxidation and identify the downstream MBD6 protein as a feasible target for developing therapies specific against TET2 mutant malignancies.

The YTHDF proteins display distinct cellular functions on m6A-modified RNA.

YTHDF proteins are main cytoplasmic 'reader' proteins of RNA N6-methyladenosine (m6A) methylation in mammals. They are largely responsible for m6A-mediated regulation in the cell cytosol by controlling both mRNA translation and degradation. Recent functional and mechanistic investigations of the YTHDF proteins revealed that these proteins have different functions to enable versatile regulation of the epitranscriptome. Their divergent functions largely originate from their different amino acid sequences in the low-complexity N termini. Consequently, they have different phase separation propensities and possess distinct post-translational modifications (PTMs). Different PTMs, subcellular localizations, and competition among partner proteins have emerged as three major mechanisms that control the functions of these YTHDF proteins. We also summarize recent progress on critical roles of these YTHDF proteins in anticancer immunity and the potential for targeting these proteins for developing new anticancer therapies.

m6A mRNA methylation by METTL14 regulates early pancreatic cell differentiation.

N6-methyladenosine (m6A) is the most abundant chemical modification in mRNA and plays important roles in human and mouse embryonic stem cell pluripotency, maintenance, and differentiation. We have recently reported that m6A is involved in the postnatal control of ß-cell function in physiological states and in type 1 and 2 diabetes. However, the precise mechanisms by which m6A acts to regulate the development of human and mouse pancreas are unexplored. Here, we show that the m6A landscape is dynamic during human pancreas development, and that METTL14, one of the m6A writer complex proteins, is essential for the early differentiation of both human and mouse pancreatic cells.

Profiling of RNA-binding protein binding sites by in situ reverse transcription-based sequencing.

RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site sequencing (ARTR-seq), which relies on in situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet crosslinking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic RNA binding by RBPs over a short period of time, as demonstrated by the profiling of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 minutes.

Zebrafish Mbd5 binds to RNA m5C and regulates histone deubiquitylation and gene expression in development metabolism and behavior.

Complex biological processes are regulated by both genetic and epigenetic programs. One class of epigenetic modifications is methylation. Evolutionarily conserved methyl-CpG-binding domain (MBD)-containing proteins are known as readers of DNA methylation. MBD5 is linked to multiple human diseases but its mechanism of action remains unclear. Here we report that the zebrafish Mbd5 does not bind to methylated DNA; but rather, it directly binds to 5-methylcytosine (m5C)-modified mRNAs and regulates embryonic development, erythrocyte differentiation, iron metabolism, and behavior. We further show that Mbd5 facilitates removal of the monoubiquitin mark at histone H2A-K119 through an interaction with the Polycomb repressive deubiquitinase (PR-DUB) complex in vivo. The direct target genes of Mbd5 are enriched with both RNA m5C and H2A-K119 ubiquitylation signals. Together, we propose that zebrafish MBD5 is an RNA m5C reader that potentially links RNA methylation to histone modification and in turn transcription regulation in vivo.

5-methylcytosine (m5C) RNA modification controls the innate immune response to virus infection by regulating type I interferons.

5-methylcytosine (m5C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m5C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m5C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant noncoding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I-mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m5C methylation controls antiviral innate immunity through modulating the m5C methylome of ncRNAs and their expression.

YTHDF2 promotes mitotic entry and is regulated by cell cycle mediators.

The N6-methyladenosine (m6A) modification regulates mRNA stability and translation. Here, we show that transcriptomic m6A modification can be dynamic and the m6A reader protein YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) promotes mRNA decay during cell cycle. Depletion of YTHDF2 in HeLa cells leads to the delay of mitotic entry due to overaccumulation of negative regulators of cell cycle such as Wee1-like protein kinase (WEE1). We demonstrate that WEE1 transcripts contain m6A modification, which promotes their decay through YTHDF2. Moreover, we found that YTHDF2 protein stability is dependent on cyclin-dependent kinase 1 (CDK1) activity. Thus, CDK1, YTHDF2, and WEE1 form a feedforward regulatory loop to promote mitotic entry. We further identified Cullin 1 (CUL1), Cullin 4A (CUL4A), damaged DNA-binding protein 1 (DDB1), and S-phase kinase-associated protein 2 (SKP2) as components of E3 ubiquitin ligase complexes that mediate YTHDF2 proteolysis. Our study provides insights into how cell cycle mediators modulate transcriptomic m6A modification, which in turn regulates the cell cycle.

Chemical Proteomic Discovery of Isotype-Selective Covalent Inhibitors of the RNA Methyltransferase NSUN2.

5-Methylcytosine (m5 C) is an RNA modification prevalent on tRNAs, where it can protect tRNAs from endonucleolytic cleavage to maintain protein synthesis. The NSUN family (NSUN1-7 in humans) of RNA methyltransferases are capable of installing the methyl group onto the C5 position of cytosines in RNA. NSUNs are implicated in a wide range of (patho)physiological processes, but selective and cell-active inhibitors of these enzymes are lacking. Here, we use cysteine-directed activity-based protein profiling (ABPP) to discover azetidine acrylamides that act as stereoselective covalent inhibitors of human NSUN2. Despite targeting a conserved catalytic cysteine in the NSUN family, the NSUN2 inhibitors show negligible cross-reactivity with other human NSUNs and exhibit good proteome-wide selectivity. We verify that the azetidine acrylamides inhibit the catalytic activity of recombinant NSUN2, but not NSUN6, and demonstrate that these compounds stereoselectively disrupt NSUN2-tRNA interactions in cancer cells, leading to a global reduction in tRNA m5 C content. Our findings thus highlight the potential to create isotype-selective and cell-active inhibitors of NSUN2 with covalent chemistry targeting a conserved catalytic cysteine.

Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling.

RNA molecules are highly compartmentalized in eukaryotic cells, with their localizations intimately linked to their functions. Despite the importance of RNA targeting, our current knowledge of the spatial organization of the transcriptome has been limited by a lack of analytical tools. In this study, we develop a chemical biology approach to label RNAs in live cells with high spatial specificity. Our method, called CAP-seq, capitalizes on light-activated, proximity-dependent photo-oxidation of RNA nucleobases, which could be subsequently enriched via affinity purification and identified by high-throughput sequencing. Using this technique, we investigate the local transcriptomes that are proximal to various subcellular compartments, including the endoplasmic reticulum and mitochondria. We discover that messenger RNAs encoding for ribosomal proteins and oxidative phosphorylation pathway proteins are highly enriched at the outer mitochondrial membrane. Due to its specificity and ease of use, CAP-seq is a generally applicable technique to investigate the spatial transcriptome in many biological systems.

MiR-195 suppression alleviates apoptosis and oxidative stress in CCl4-induced ALI in mice by targeting Pim-1.

BACKGROUND: Acute liver injury (ALI) is associated with the oxidative stress and apoptosis in liver. Recent studies have shown that miR-195, a critical member of miR-15 family, has modulated the apoptosis in various organic diseases. However, it is elusive whether miR-195 regulation exert a hepatic ameliorative effect on ALI by the suppression of apoptosis and oxidative stress levels. We aimed to explore the regulated role of miR-195 in acute liver injury via the current study. METHODS: C57BL/6 J mice (male, seven-week, 18-20 g) were administrated intraperitoneal injection with tetrachloromethane (CCl4) to induce ALI. miR-195 inhibitor or mimics loaded in lentivirus vectors (miR-195 INH or MMC) and Pim-1 loaded in Adeno-associated viral vectors (AAV-Pim-1) were respectively delivered into mouse tail intravenous to establish silence or overexpression of miR-195 and overexpression of Pim-1. Western blotting, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), enzyme linked immunosorbent assay (ELISA) technique, Immunohistochemistry (IHC) and Hematoxylin-eosin (H&E) staining were conducted to measure miR-195 and Pim-1 expression, apoptosis and oxidative stress levels, histological and functional change. RESULTS: We found that the expression of miR-195 markedly increased in CCl4-induced ALI. Besides, we demonstrated that the silence of miR-195 attenuated the apoptosis and oxidative stress via up-regulating Pim-1 in CCl4-induced ALI. Moreover, the inhibition of miR-195 protected the integrity and function of liver tissue. CONCLUSIONS: The above results showed that the suppression of miR-195 ameliorated ALI through inhibiting apoptosis and oxidative stress via targeting Pim-1. Our research provided a novel scheme that the miR-195 modulation in process of ALI may be an effective therapy method and verifies a promising target for diagnostic and therapeutic strategy of miRNAs.

YTHDF1 mediates translational control by m6A mRNA methylation in adaptation to environmental challenges.

Animals adapt to environmental challenges with long-term changes at the behavioral, circuit, cellular, and synaptic levels which often require new protein synthesis. The discovery of reversible N6-methyladenosine (m6A) modifications of mRNA has revealed an important layer of post-transcriptional regulation which affects almost every phase of mRNA metabolism and therefore translational control. Many in vitro and in vivo studies have demonstrated the significant role of m6A in cell differentiation and survival, but its role in adult neurons is understudied. We used cell-type specific gene deletion of Mettl14, which encodes one of the subunits of the m6A methyltransferase, and Ythdf1, which encodes one of the cytoplasmic m6A reader proteins, in dopamine D1 receptor expressing or D2 receptor expressing neurons. Mettl14 or Ythdf1 deficiency blunted responses to environmental challenges at the behavioral, cellular, and molecular levels. In three different behavioral paradigms, gene deletion of either Mettl14 or Ythdf1 in D1 neurons impaired D1-dependent learning, whereas gene deletion of either Mettl14 or Ythdf1 in D2 neurons impaired D2-dependent learning. At the cellular level, modulation of D1 and D2 neuron firing in response to changes in environments was blunted in all three behavioral paradigms in mutant mice. Ythdf1 deletion resembled impairment caused by Mettl14 deletion in a cell type-specific manner, suggesting YTHDF1 is the main mediator of the functional consequences of m6A mRNA methylation in the striatum. At the molecular level, while striatal neurons in control mice responded to elevated cAMP by increasing de novo protein synthesis, striatal neurons in Ythdf1 knockout mice didn't. Finally, boosting dopamine release by cocaine drastically increased YTHDF1 binding to many mRNA targets in the striatum, especially those that encode structural proteins, suggesting the initiation of long-term neuronal and/or synaptic structural changes. While the m6A-YTHDF1 pathway has similar functional significance at cellular level, its cell type specific deficiency in D1 and D2 neurons often resulted in contrasting behavioral phenotypes, allowing us to cleanly dissociate the opposing yet cooperative roles of D1 and D2 neurons.

New horizons of regulatory RNA.

Genetic information flows from DNA to protein through RNA in the central dogma. Different RNA species are known to accomplish essential tasks of protein encoding (mRNAs), amino acid loading (tRNAs), and translation machinery assembly (rRNAs). However, on top of these well-known roles, RNAs are central to various cellular regulatory pathways. Here we summarize newly emerging regulatory functions of RNA, specifically focusing on regulations through RNA modifications, RNP granules, and chromatin-associated regulatory RNA. In addition to being an essential building block of the central dogma, RNA can be critical to the regulation of many cellular processes.

The mechanism underlying redundant functions of the YTHDF proteins.

The YTH N6-methyladenosine RNA binding proteins (YTHDFs) mediate the functional effects of N6-methyladenosine (m6A) on RNA. Recently, a report proposed that all YTHDFs work redundantly to facilitate RNA decay, raising questions about the exact functions of individual YTHDFs, especially YTHDF1 and YTHDF2. We show that YTHDF1 and YTHDF2 differ in their low-complexity domains (LCDs) and exhibit different behaviors in condensate formation and subsequent physiological functions. Biologically, we also find that the global stabilization of RNA after depletion of all YTHDFs is driven by increased P-body formation and is not strictly m6A dependent.

m 6 A mRNA Methylation Regulates Early Pancreatic ß-Cell Differentiation.

N 6 -methyladenosine (m 6 A) is the most abundant chemical modification in mRNA, and plays important roles in human and mouse embryonic stem cell pluripotency, maintenance, and differentiation. We have recently reported, for the first time, the role of m 6 A in the postnatal control of ß-cell function in physiological states and in Type 1 and 2 Diabetes. However, the precise mechanisms by which m 6 A acts to regulate the development of human and mouse ß-cells are unexplored. Here, we show that the m 6 A landscape is dynamic during human pancreas development, and that METTL14, one of the m 6 A writer complex proteins, is essential for the early differentiation of both human and mouse ß-cells.

O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins.

N6-methyladenosine (m6A) is the most abundant internal mRNA nucleotide modification in mammals, regulating critical aspects of cell physiology and differentiation. The YTHDF proteins are the primary readers of m6A modifications and exert physiological functions of m6A in the cytosol. Elucidating the regulatory mechanisms of YTHDF proteins is critical to understanding m6A biology. Here we report a mechanism that protein post-translational modifications control the biological functions of the YTHDF proteins. We find that YTHDF1 and YTHDF3, but not YTHDF2, carry high levels of nutrient-sensing O-GlcNAc modifications. O-GlcNAcylation attenuates the translation-promoting function of YTHDF1 and YTHDF3 by blocking their interactions with proteins associated with mRNA translation. We further demonstrate that O-GlcNAc modifications on YTHDF1 and YTHDF3 regulate the assembly, stability and disassembly of stress granules to enable better recovery from stress. Therefore, our results discover an important regulatory pathway of YTHDF functions, adding an additional layer of complexity to the post-transcriptional regulation function of mRNA m6A.

Exon architecture controls mRNA m6A suppression and gene expression.

N6-methyladenosine (m6A) is the most abundant messenger RNA (mRNA) modification and plays crucial roles in diverse physiological processes. Using a massively parallel assay for m6A (MPm6A), we discover that m6A specificity is globally regulated by suppressors that prevent m6A deposition in unmethylated transcriptome regions. We identify exon junction complexes (EJCs) as m6A suppressors that protect exon junction-proximal RNA within coding sequences from methylation and regulate mRNA stability through m6A suppression. EJC suppression of m6A underlies multiple global characteristics of mRNA m6A specificity, with the local range of EJC protection sufficient to suppress m6A deposition in average-length internal exons but not in long internal and terminal exons. EJC-suppressed methylation sites colocalize with EJC-suppressed splice sites, which suggests that exon architecture broadly determines local mRNA accessibility to regulatory complexes.

Metabolic incorporation of electron-rich ribonucleosides enhances APEX-seq for profiling spatially restricted nascent transcriptome.

The spatial arrangement of newly synthesized transcriptome in eukaryotic cells underlies various biological processes including cell proliferation and differentiation. In this study, we combine metabolic incorporation of electron-rich ribonucleosides (e.g., 6-thioguanosine and 4-thiouridine) with a peroxidase-mediated proximity-dependent RNA labeling technique (APEX-seq) to develop a sensitive method, termed MERR APEX-seq, for selectively profiling newly transcribed RNAs at specific subcellular locations in live cells. We demonstrate that MERR APEX-seq is 20-fold more efficient than APEX-seq and offers both high spatial specificity and high coverage in mitochondrial matrix. At the ER membrane, 91% of the transcripts captured by MERR APEX-seq encode for secretory pathway proteins, thus demonstrating the high spatial specificity of MERR APEX-seq in open subcellular compartments. Application of MERR APEX-seq to the nuclear lamina of human cells reveals a local transcriptome of 1,012 RNAs, many of which encode for nuclear proteins involved in histone modification, chromosomal structure maintenance, and RNA processing.