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The methyltransferase METTL3 promotes the leukaemic state in acute myeloid leukaemia (AML) by catalysing the m 6 A RNA modification through its recruitment on the transcription start sites of AML-associated genes. METTL3 supports tumour growth N 6 -methyladenosine (m 6 A) is an RNA modification in coding and non-coding RNAs that is catalysed by the METTL3–METTL14 methyltransferase complex and affects various aspects of RNA metabolism such as splicing, translation and degradation. Here, Tony Kouzarides, George Vassiliou and colleagues perform a CRISPR–Cas9 lethality screen and identify METTL3 as a gene that is essential for the growth of acute myeloid leukaemia (AML) cells. METTL3 associates with chromatin and is recruited to the promoters of active genes that are required for AML. At these gene promoters, METTL3 catalyses m 6 A within the coding region of the associated mRNA transcripts and enhances their translation by relieving ribosome stalling. These results indicate that METTL3 has an oncogenic role in AML and is a potential therapeutic target. N 6 -methyladenosine (m 6 A) is an abundant internal RNA modification in both coding 1 and non-coding RNAs 2 , 3 that is catalysed by the METTL3–METTL14 methyltransferase complex 4 . However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site 5 , and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m 6 A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia.

Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT 2A receptor activation mediates psychedelic effects, prototypical psychedelics activate both 5-HT 2A -Gq/11 and β-arrestin2 transducers, making their respective roles unclear. To elucidate this, we develop a series of 5-HT 2A -selective ligands with varying Gq efficacies, including β-arrestin-biased ligands. We show that 5-HT 2A -Gq but not 5-HT 2A -β-arrestin2 recruitment efficacy predicts psychedelic potential, assessed using head-twitch response (HTR) magnitude in male mice. We further show that disrupting Gq-PLC signaling attenuates the HTR and a threshold level of Gq activation is required to induce psychedelic-like effects, consistent with the fact that certain 5-HT 2A partial agonists (e.g., lisuride) are non-psychedelic. Understanding the role of 5-HT 2A Gq-efficacy in psychedelic-like psychopharmacology permits rational development of non-psychedelic 5-HT 2A agonists. We also demonstrate that β-arrestin-biased 5-HT 2A receptor agonists block psychedelic effects and induce receptor downregulation and tachyphylaxis. Overall, 5-HT 2A receptor Gq-signaling can be fine-tuned to generate ligands distinct from classical psychedelics. Serotonin 5-HT 2A receptor signaling mechanisms associated with predicting psychedelic potential remain elusive. Using 5-HT 2A -selective β-arrestin-biased ligands, here the authors show that a threshold level of 5-HT 2A -Gq efficacy and not β-arrestin recruitment is associated with psychedelic potential.

Gastric cancer (GC), one of the most common malignant tumors in the world, exhibits a rapid metastasis rate and causes high mortality. Diagnostic markers and potential therapeutic targets for GCs are urgently needed. Here we show that Actin-like protein 6 A (ACTL6A), encoding an SWI/SNF subunit, is highly expressed in GCs. ACTL6A is found to be critical for regulating the glutathione (GSH) metabolism pathway because it upregulates γ-glutamyl-cysteine ligase catalytic subunit (GCLC) expression, thereby reducing reactive oxygen species (ROS) levels and inhibiting ferroptosis, a regulated form of cell death driven by the accumulation of lipid-based ROS. Mechanistic studies show that ACTL6A upregulates GCLC as a cotranscription factor with Nuclear factor (erythroid-derived 2)-like 2 (NRF2) and that the hydrophobic region of ACTL6A plays an important role. Our data highlight the oncogenic role of ACTL6A in GCs and indicate that inhibition of ACTL6A or GCLC could be a potential treatment strategy for GCs. Diagnostic markers and therapeutic targets for gastric cancer are in urgent need. Here, the authors 2 find elevated expression of ACTL6A in gastric cancer promotes glutathione synthesis by regulating 3 GCLC expression to suppress ferroptosis.

Because of their small size, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing. CRISPR-Cas12f nucleases can be effectively packaged into AAVs for gene therapy, but a systematic evaluation of editing outcomes is lacking. Here the authors perform a comprehensive assessment of 4 Cas12f proteins and compare to Cas9 and two Cas12a proteins at a number of sites.

The mechanisms underlying gene repression and silencers are poorly understood. Here we investigate the hypothesis that H3K27me3-rich regions of the genome, defined from clusters of H3K27me3 peaks, may be used to identify silencers that can regulate gene expression via proximity or looping. We find that H3K27me3-rich regions are associated with chromatin interactions and interact preferentially with each other. H3K27me3-rich regions component removal at interaction anchors by CRISPR leads to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. Cells with H3K27me3-rich regions knockout also show changes in phenotype associated with cell identity, and altered xenograft tumor growth. Finally, we observe that H3K27me3-rich regions-associated genes and long-range chromatin interactions are susceptible to H3K27me3 depletion. Our results characterize H3K27me3-rich regions and their mechanisms of functioning via looping. Mechanisms underlying gene repression and silencers remain poorly understood. Here the authors investigate the role of H3K27me3-rich regions in the genome, as defined from clusters of H3K27me3 peaks, in regulating gene expression via looping.

SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Although population average structure models of the genome were recently reported, there is little experimental data on native structural ensembles, and most structures lack functional characterization. Here we report secondary structure heterogeneity of the entire SARS-CoV-2 genome in two lines of infected cells at single nucleotide resolution. Our results reveal alternative RNA conformations across the genome and at the critical frameshifting stimulation element (FSE) that are drastically different from prevailing population average models. Importantly, we find that this structural ensemble promotes frameshifting rates much higher than the canonical minimal FSE and similar to ribosome profiling studies. Our results highlight the value of studying RNA in its full length and cellular context. The genomic structures detailed here lay groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics. Lan et al. report RNA structure ensembles across the entire SARSCoV-2 genome in infected human cells at single nucleotide resolution. They find alternative RNA conformations critical for promoting near-native frameshifting rates in ORF1ab.

Reversible post-translational modifications represent a mechanism to control tumor metabolism. Here we show that mitochondrial Sirtuin5 (SIRT5), which mediates lysine desuccinylation, deglutarylation, and demalonylation, plays a role in colorectal cancer (CRC) glutamine metabolic rewiring. Metabolic profiling identifies that deletion of SIRT5 causes a marked decrease in 13 C-glutamine incorporation into tricarboxylic-acid (TCA) cycle intermediates and glutamine-derived non-essential amino acids. This reduces the building blocks required for rapid growth. Mechanistically, the direct interaction between SIRT5 and glutamate dehydrogenase 1 (GLUD1) causes deglutarylation and functional activation of GLUD1, a critical regulator of cellular glutaminolysis. Consistently, GLUD1 knockdown diminishes SIRT5-induced proliferation, both in vivo and in vitro. Clinically, overexpression of SIRT5 is significantly correlated with poor prognosis in CRC. Thus, SIRT5 supports the anaplerotic entry of glutamine into the TCA cycle in malignant phenotypes of CRC via activating GLUD1. Tumour metabolism can be controlled through post-translational modifications. Here the authors show that Sirtuin5 promotes glutaminolysis in colorectal cancer cells via glutamate dehydrogenase-1, a critical regulator of glutaminolysis, inducing its deglutarylation and functional activation.

The targeting range of the CRISPR endonuclease Cpf1 is increased three-fold by molecular engineering. The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells 1 , 2 , 3 , 4 , 5 , 6 , 7 . However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS 7 indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.

Mesenchymal stem cells (MSCs) possess potent immunomodulatory activity and have been extensively investigated for their therapeutic potential in treating inflammatory disorders. However, the mechanisms underlying the immunosuppressive function of MSCs are not fully understood, hindering the development of standardized MSC-based therapies for clinical use. In this study, we profile the single-cell transcriptomes of MSCs isolated from adipose tissue (AD), bone marrow (BM), placental chorionic membrane (PM), and umbilical cord (UC). Our results demonstrate that MSCs undergo a progressive aging process and that the cellular senescence state influences their immunosuppressive activity by downregulating PD-L1 expression. Through integrated analysis of single-cell transcriptomic and proteomic data, we identify GATA2 as a regulator of MSC senescence and PD-L1 expression. Overall, our findings highlight the roles of cell aging and PD-L1 expression in modulating the immunosuppressive efficacy of MSCs and implicating perinatal MSC therapy for clinical applications in inflammatory disorders. Mesenchymal stem cells (MSC) are used for immunosuppressive therapy and a uniform source or heterogeneity characterisation is needed. Here the authors use multi-omics to compare human MSC from different sources and ages of donors and show differences in gene expression and immunosuppressive function.

Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRAS G12D , presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRAS G12D over KRAS WT . While crystal structures reveal few discernible differences in TCR interactions with KRAS WT versus KRAS G12D , thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRAS G12D . Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen. Cancers often harbor mutations in genes encoding important regulatory proteins, but therapeutic targeting of these molecules proves difficult due to their high structural similarity to their non-mutated counterpart. Here authors show the engineering of T cell engaging bispecific protein able to selectively target cancer cells with a high-frequency mutation in the KRAS oncogene.