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5-methylcytosine （m5C） is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of trans- lation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specif- ically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF＇s nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of AL- YREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not meth- yltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.

Chemical modifications of RNA have key roles in many biological processes 1 – 3 . N 7 -methylguanosine (m 7 G) is required for integrity and stability of a large subset of tRNAs 4 – 7 . The methyltransferase 1–WD repeat-containing protein 4 (METTL1–WDR4) complex is the methyltransferase that modifies G46 in the variable loop of certain tRNAs, and its dysregulation drives tumorigenesis in numerous cancer types 8 – 14 . Mutations in WDR4 cause human developmental phenotypes including microcephaly 15 – 17 . How METTL1–WDR4 modifies tRNA substrates and is regulated remains elusive 18 . Here we show,  through structural, biochemical and cellular studies of human METTL1–WDR4, that WDR4 serves as a scaffold for METTL1 and the tRNA T-arm. Upon tRNA binding, the αC region of METTL1 transforms into a helix, which together with the α6 helix secures both ends of the tRNA variable loop. Unexpectedly, we find that the predicted disordered N-terminal region of METTL1 is part of the catalytic pocket and essential for methyltransferase activity. Furthermore, we reveal that S27 phosphorylation in the METTL1 N-terminal region inhibits methyltransferase activity by locally disrupting the catalytic centre. Our results provide a molecular understanding of tRNA substrate recognition and phosphorylation-mediated regulation of METTL1–WDR4, and reveal the presumed disordered N-terminal region of METTL1 as a nexus of methyltransferase activity. Structures of the human METTL1–WDR4 complex are revealed, providing molecular insights into substrate recognition, modification and catalytic regulation by the N 7 -methylguanosine methyltransferase complex.

Cysteine palmitoylation (S-palmitoylation) is a reversible post-translational modification that is installed by the DHHC family of palmitoyltransferases and is reversed by several acyl protein thioesterases 1 , 2 . Although thousands of human proteins are known to undergo S-palmitoylation, how this modification is regulated to modulate specific biological functions is poorly understood. Here we report that the key T helper 17 (T H 17) cell differentiation stimulator, STAT3 3 , 4 , is subject to reversible S-palmitoylation on cysteine 108. DHHC7 palmitoylates STAT3 and promotes its membrane recruitment and phosphorylation. Acyl protein thioesterase 2 (APT2, also known as LYPLA2) depalmitoylates phosphorylated STAT3 (p-STAT3) and enables it to translocate to the nucleus. This palmitoylation–depalmitoylation cycle enhances STAT3 activation and promotes T H 17 cell differentiation; perturbation of either palmitoylation or depalmitoylation negatively affects T H 17 cell differentiation. Overactivation of T H 17 cells is associated with several inflammatory diseases, including inflammatory bowel disease (IBD). In a mouse model, pharmacological inhibition of APT2 or knockout of Zdhhc7 —which encodes DHHC7—relieves the symptoms of IBD. Our study reveals not only a potential therapeutic strategy for the treatment of IBD but also a model through which S-palmitoylation regulates cell signalling, which might be broadly applicable for understanding the signalling functions of numerous S-palmitoylation events. The dynamic and reversible S-palmitoylation of the transcription factor STAT3 enhances its activation and promotes the differentiation of T H 17 cells.

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes 1 – 6 ; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N -acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N -acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh- expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network. We find that bile salt hydrolase N -acyltransferase activity can form bacterial bile acid amidates that are positively correlated with the colonization of gut bacteria that assist in the regulation of the bile acid metabolic network.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties 1 – 4 . Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health. Acyltransferase activity of the enzyme bile salt hydrolase is identified and shown to mediate microbial bile acid conjugation, diversifying the bile acid pool and expanding their role in gut physiology.

Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N -acetylglucosamine (GlcNAc) units 1 . The key reactions of chitin biosynthesis are catalysed by chitin synthase 2 – 4 , a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae ( Ps Chs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a ‘gate lock’ that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis. Using cryo-electron microscopy, the directional multiple step mechanism of chitin biosynthesis is revealed.

Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. We additionally observe that the HIV-1 promoter displays stochastic fluctuations on two time scales, which we refer to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. We propose that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells. HIV-1 viral gene expression stochastically switches between active and inactive states. Here, using improved single molecule RNA microscopy, the authors show that HIV-1 RNA stochastic transcription is achieved by groups of closely spaced polymerases, and is regulated by Mediator and TBP at different time scales.

The histone acetyl transferase KAT2A (also known as GCN5) can also catalyse histone succinylation, with the α-KGDH complex providing a local source of succinyl-CoA. GCN5 in histone succyinylation Succinylation of lysines has been identified as a post-translational modification of histones, but the enzymes that deposit it and its functional consequences are unknown. Here, Zhimin Lu and colleagues find that GCN5, a known histone acetyl transferase, can also catalyse histone succinylation. GCN5 interacts with nuclear succinyl-CoA and with the enzyme α-ketoglutarate dehydrogenase (α-KGDH), which generates a local source of succinyl-CoA. The complex of GCN5 and α-KGDH can regulate histone H3K79 succinylation around transcription start sites and affect gene expression. The authors also show that a reduction in H3K79 succinylation is associated with inhibited proliferation of tumour cells in mice. Histone modifications, such as the frequently occurring lysine succinylation 1 , 2 , are central to the regulation of chromatin-based processes. However, the mechanism and functional consequences of histone succinylation are unknown. Here we show that the α-ketoglutarate dehydrogenase (α-KGDH) complex is localized in the nucleus in human cell lines and binds to lysine acetyltransferase 2A (KAT2A, also known as GCN5) in the promoter regions of genes. We show that succinyl-coenzyme A (succinyl-CoA) binds to KAT2A. The crystal structure of the catalytic domain of KAT2A in complex with succinyl-CoA at 2.3 Å resolution shows that succinyl-CoA binds to a deep cleft of KAT2A with the succinyl moiety pointing towards the end of a flexible loop 3, which adopts different structural conformations in succinyl-CoA-bound and acetyl-CoA-bound forms. Site-directed mutagenesis indicates that tyrosine 645 in this loop has an important role in the selective binding of succinyl-CoA over acetyl-CoA. KAT2A acts as a succinyltransferase and succinylates histone H3 on lysine 79, with a maximum frequency around the transcription start sites of genes. Preventing the α-KGDH complex from entering the nucleus, or expression of KAT2A(Tyr645Ala), reduces gene expression and inhibits tumour cell proliferation and tumour growth. These findings reveal an important mechanism of histone modification and demonstrate that local generation of succinyl-CoA by the nuclear α-KGDH complex coupled with the succinyltransferase activity of KAT2A is instrumental in histone succinylation, tumour cell proliferation, and tumour development.

Epitranscriptome modifications are required for structure and function of RNA and defects in these pathways have been associated with human disease. Here we identify the RNA target for the previously uncharacterized 5-methylcytosine (m 5 C) methyltransferase NSun3 and link m 5 C RNA modifications with energy metabolism. Using whole-exome sequencing, we identified loss-of-function mutations in NSUN3 in a patient presenting with combined mitochondrial respiratory chain complex deficiency. Patient-derived fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of NSun3. We show that NSun3 is required for deposition of m 5 C at the anticodon loop in the mitochondrially encoded transfer RNA methionine (mt-tRNA Met ). Further, we demonstrate that m 5 C deficiency in mt-tRNA Met results in the lack of 5-formylcytosine (f 5 C) at the same tRNA position. Our findings demonstrate that NSUN3 is necessary for efficient mitochondrial translation and reveal that f 5 C in human mitochondrial RNA is generated by oxidative processing of m 5 C. The post-transcriptional 5-methylcytosine (m 5 C) modification occurs in a wide range of nuclear-encoded RNAs. Here the authors identify the mitochondrial tRNA-Met as a target for the m 5 C methyltransferase NSun3—found mutated in a mitochondrial disease patient—and link mitochondrial tRNA modifications with energy metabolism.

Phosphatidylserine (PtdSer) is a phospholipid that is abundant in eukaryotic plasma membranes. An ATP-dependent enzyme called flippase normally keeps PtdSer inside the cell, but PtdSer is exposed by the action of scramblase on the cell’s surface in biological processes such as apoptosis and platelet activation. Once exposed to the cell surface, PtdSer acts as an ‘eat me’ signal on dead cells, and creates a scaffold for blood-clotting factors on activated platelets. The molecular identities of the flippase and scramblase that work at plasma membranes have long eluded researchers. Indeed, their identity as well as the mechanism of the PtdSer exposure to the cell surface has only recently been revealed. Here, we describe how PtdSer is exposed in apoptotic cells and in activated platelets, and discuss PtdSer exposure in other biological processes.