RNA quality control factors nucleate Clr4/SUV39H and trigger constitutive heterochromatin assembly.

In eukaryotes, the Suv39 family of proteins tri-methylate lysine 9 of histone H3 (H3K9me) to form constitutive heterochromatin. However, how Suv39 proteins are nucleated at heterochromatin is not fully described. In the fission yeast, current models posit that Argonaute1-associated small RNAs (sRNAs) nucleate the sole H3K9 methyltransferase, Clr4/SUV39H, to centromeres. Here, we show that in the absence of all sRNAs and H3K9me, the Mtl1 and Red1 core (MTREC)/PAXT complex nucleates Clr4/SUV39H at a heterochromatic long noncoding RNA (lncRNA) at which the two H3K9 deacetylases, Sir2 and Clr3, also accumulate by distinct mechanisms. Iterative cycles of H3K9 deacetylation and methylation spread Clr4/SUV39H from the nucleation center in an sRNA-independent manner, generating a basal H3K9me state. This is acted upon by the RNAi machinery to augment and amplify the Clr4/H3K9me signal at centromeres to establish heterochromatin. Overall, our data reveal that lncRNAs and RNA quality control factors can nucleate heterochromatin and function as epigenetic silencers in eukaryotes.

Hematopoietic stem cell niche generation and maintenance are distinguishable by an epitranscriptomic program.

The niche is typically considered as a pre-established structure sustaining stem cells. Therefore, the regulation of its formation remains largely unexplored. Whether distinct molecular mechanisms control the establishment versus maintenance of a stem cell niche is unknown. To address this, we compared perinatal and adult bone marrow mesenchymal stromal cells (MSCs), a key component of the hematopoietic stem cell (HSC) niche. MSCs exhibited enrichment in genes mediating m6A mRNA methylation at the perinatal stage and downregulated the expression of Mettl3, the m6A methyltransferase, shortly after birth. Deletion of Mettl3 from developing MSCs but not osteoblasts led to excessive osteogenic differentiation and a severe HSC niche formation defect, which was significantly rescued by deletion of Klf2, an m6A target. In contrast, deletion of Mettl3 from MSCs postnatally did not affect HSC niche. Stem cell niche generation and maintenance thus depend on divergent molecular mechanisms, which may be exploited for regenerative medicine.

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis.

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

A Unified Model for the Function of YTHDF Proteins in Regulating m6A-Modified mRNA.

N6-methyladenosine (m6A) is the most abundant mRNA nucleotide modification and regulates critical aspects of cellular physiology and differentiation. m6A is thought to mediate its effects through a complex network of interactions between different m6A sites and three functionally distinct cytoplasmic YTHDF m6A-binding proteins (DF1, DF2, and DF3). In contrast to the prevailing model, we show that DF proteins bind the same m6A-modified mRNAs rather than different mRNAs. Furthermore, we find that DF proteins do not induce translation in HeLa cells. Instead, the DF paralogs act redundantly to mediate mRNA degradation and cellular differentiation. The ability of DF proteins to regulate stability and differentiation becomes evident only when all three DF paralogs are depleted simultaneously. Our study reveals a unified model of m6A function in which all m6A-modified mRNAs are subjected to the combined action of YTHDF proteins in proportion to the number of m6A sites.

Evolutionary Persistence of DNA Methylation for Millions of Years after Ancient Loss of a De Novo Methyltransferase.

Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 mya. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 million years through a process analogous to Darwinian evolution of the genome.

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex.

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.

METTL13 Methylation of eEF1A Increases Translational Output to Promote Tumorigenesis.

Increased protein synthesis plays an etiologic role in diverse cancers. Here, we demonstrate that METTL13 (methyltransferase-like 13) dimethylation of eEF1A (eukaryotic elongation factor 1A) lysine 55 (eEF1AK55me2) is utilized by Ras-driven cancers to increase translational output and promote tumorigenesis in vivo. METTL13-catalyzed eEF1A methylation increases eEF1A's intrinsic GTPase activity in vitro and protein production in cells. METTL13 and eEF1AK55me2 levels are upregulated in cancer and negatively correlate with pancreatic and lung cancer patient survival. METTL13 deletion and eEF1AK55me2 loss dramatically reduce Ras-driven neoplastic growth in mouse models and in patient-derived xenografts (PDXs) from primary pancreatic and lung tumors. Finally, METTL13 depletion renders PDX tumors hypersensitive to drugs that target growth-signaling pathways. Together, our work uncovers a mechanism by which lethal cancers become dependent on the METTL13-eEF1AK55me2 axis to meet their elevated protein synthesis requirement and suggests that METTL13 inhibition may constitute a targetable vulnerability of tumors driven by aberrant Ras signaling.

The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.

Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.

Temporal Control of Mammalian Cortical Neurogenesis by m6A Methylation.

N6-methyladenosine (m6A), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether m6A regulates mammalian brain development is unknown. Here, we show that m6A depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages. m6A depletion by Mettl3 knockdown also leads to a prolonged cell cycle and maintenance of radial glia cells. m6A sequencing of embryonic mouse cortex reveals enrichment of mRNAs related to transcription factors, neurogenesis, the cell cycle, and neuronal differentiation, and m6A tagging promotes their decay. Further analysis uncovers previously unappreciated transcriptional prepatterning in cortical neural stem cells. m6A signaling also regulates human cortical neurogenesis in forebrain organoids. Comparison of m6A-mRNA landscapes between mouse and human cortical neurogenesis reveals enrichment of human-specific m6A tagging of transcripts related to brain-disorder risk genes. Our study identifies an epitranscriptomic mechanism in heightened transcriptional coordination during mammalian cortical neurogenesis.

Structural Basis of Mitochondrial Transcription Initiation.

Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria.

Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach.

5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.

DDX21: The link between m6A and R-loops.

In this issue of Molecular Cell, Hao et al.1 demonstrate that the RNA helicase DDX21 recruits the m6A methyltransferase complex to R-loops, ensuring proper transcription termination and genome stability.

Nuclear retention coupled with sequential polyadenylation dictates post-transcriptional m6A modification in the nucleus.

N6-methyladenosine (m6A) modification is deemed to be co-transcriptionally installed on pre-mRNAs, thereby influencing various downstream RNA metabolism events. However, the causal relationship between m6A modification and RNA processing is often unclear, resulting in premature or even misleading generalizations on the function of m6A modification. Here, we develop 4sU-coupled m6A-level and isoform-characterization sequencing (4sU-m6A-LAIC-seq) and 4sU-GLORI to quantify the m6A levels for both newly synthesized and steady-state RNAs at transcript and single-base-resolution levels, respectively, which enable dissecting the relationship between m6A modification and alternative RNA polyadenylation. Unexpectedly, our results show that many m6A addition events occur post-transcriptionally, especially on transcripts with high m6A levels. Importantly, we find higher m6A levels on shorter 3' UTR isoforms, which likely result from sequential polyadenylation of longer 3' UTR isoforms with prolonged nuclear dwelling time. Therefore, m6A modification can also take place post-transcriptionally to intimately couple with other key RNA metabolism processes to establish and dynamically regulate epi-transcriptomics in mammalian cells.

METTL17 is an Fe-S cluster checkpoint for mitochondrial translation.

Friedreich's ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.

RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release.

Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.

DDX21 mediates co-transcriptional RNA m6A modification to promote transcription termination and genome stability.

N6-methyladenosine (m6A) is a crucial RNA modification that regulates diverse biological processes in human cells, but its co-transcriptional deposition and functions remain poorly understood. Here, we identified the RNA helicase DDX21 with a previously unrecognized role in directing m6A modification on nascent RNA for co-transcriptional regulation. DDX21 interacts with METTL3 for co-recruitment to chromatin through its recognition of R-loops, which can be formed co-transcriptionally as nascent transcripts hybridize onto the template DNA strand. Moreover, DDX21's helicase activity is needed for METTL3-mediated m6A deposition onto nascent RNA following recruitment. At transcription termination regions, this nexus of actions promotes XRN2-mediated termination of RNAPII transcription. Disruption of any of these steps, including the loss of DDX21, METTL3, or their enzymatic activities, leads to defective termination that can induce DNA damage. Therefore, we propose that the R-loop-DDX21-METTL3 nexus forges the missing link for co-transcriptional modification of m6A, coordinating transcription termination and genome stability.

m6A modification controls the innate immune response to infection by targeting type I interferons.

N6-methyladenosine (m6A) is the most common mRNA modification. Recent studies have revealed that depletion of m6A machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the m6A 'writer' METTL3 or 'reader' YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was m6A modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that m6A-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role m6A serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.

RNA m6A methylation across the transcriptome.

Since the early days of foundational studies of nucleic acids, many chemical moieties have been discovered to decorate RNA and DNA in diverse organisms. In mammalian cells, one of these chemical modifications, N6-methyl adenosine (m6A), is unique in a way that it is highly abundant not only on RNA polymerase II (RNAPII) transcribed, protein-coding transcripts but also on non-coding RNAs, such as ribosomal RNAs and snRNAs, mediated by distinct, evolutionarily conserved enzymes. Here, we review RNA m6A modification in the light of the recent appreciation of nuclear roles for m6A in regulating chromatin states and gene expression, as well as the recent discoveries of the evolutionarily conserved methyltransferases, which catalyze methylation of adenosine on diverse sets of RNAs. Considering that the substrates of these enzymes are involved in many important biological processes, this modification warrants further research to understand the molecular mechanisms and functions of m6A in health and disease.

N6-methyladenosine in 7SK small nuclear RNA underlies RNA polymerase II transcription regulation.

N6-methyladenosine (m6A) modifications play crucial roles in RNA metabolism. How m6A regulates RNA polymerase II (RNA Pol II) transcription remains unclear. We find that 7SK small nuclear RNA (snRNA), a regulator of RNA Pol II promoter-proximal pausing, is highly m6A-modified in non-small cell lung cancer (NSCLC) cells. In A549 cells, we identified eight m6A sites on 7SK and discovered methyltransferase-like 3 (METTL3) and alkB homolog 5 (ALKBH5) as the responsible writer and eraser. When the m6A-7SK is specifically erased by a dCasRx-ALKBH5 fusion protein, A549 cell growth is attenuated due to reduction of RNA Pol II transcription. Mechanistically, removal of m6A leads to 7SK structural rearrangements that facilitate sequestration of the positive transcription elongation factor b (P-TEFb) complex, which results in reduction of serine 2 phosphorylation (Ser2P) in the RNA Pol II C-terminal domain and accumulation of RNA Pol II in the promoter-proximal region. Taken together, we uncover that m6A modifications of a non-coding RNA regulate RNA Pol II transcription and NSCLC tumorigenesis.

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.