Methyltransferase Domain Research Articles

Suppression of human STAT2 by ZIKV NS5

Abstract The infection of ZIKA virus (ZIKV), a mosquito-transmitted flavivirus, has been shown to cause congenital disease in the context of infection during pregnancy in its 2015 outbreak. To establish the infection, ZIKV must suppress host interferon (IFN) response. Previous studies have shown that ZIKV utilizes its non-structural NS5 protein to trigger the human STAT2, a critical mediator to boost host interferon (IFN) response, for the proteasome-dependent degradation. However, the underlying mechanisms of viral NS5 and hSTAT2 remain elusive largely due to the lack of atom level conformational information. Here, we report the structure of hSTAT2 in complex with ZIKV NS5. hSTAT2, with the SH2 domain occluded by the C-terminal tyrosine-phosphorylation (pY)-tail segment, inserts the coiled-coil domain into the cleft between the methyltransferase and RdRP domains of NS5 in a manner that would occlude the association of hSTAT2 with cellular regulatory protein IRF9. Disruption of the ZIKV NS5 – hSTAT2 association in cells compromises the NS5-mediated hSTAT2 degradation and IFN responses. This study provides a basis for understanding the functional antagonism between viral antagonists and STAT2.

Supramolecular arrangement of the full-length Zika virus NS5.

Zika virus (ZIKV), a member of the Flaviviridae family, has emerged as a major public health threat, since ZIKV infection has been connected to microcephaly and other neurological disorders. Flavivirus genome replication is driven by NS5, an RNA-dependent RNA polymerase (RdRP) that also contains a N-terminal methyltransferase domain essential for viral mRNA capping. Given its crucial roles, ZIKV NS5 has become an attractive antiviral target. Here, we have used integrated structural biology approaches to characterize the supramolecular arrangement of the full-length ZIKV NS5, highlighting the assembly and interfaces between NS5 monomers within a dimeric structure, as well as the dimer-dimer interactions to form higher order fibril-like structures. The relative orientation of each monomer within the dimer provides a model to explain the coordination between MTase and RdRP domains across neighboring NS5 molecules and mutational studies underscore the crucial role of the MTase residues Y25, K28 and K29 in NS5 dimerization. The basic residue K28 also participates in GTP binding and competition experiments indicate that NS5 dimerization is disrupted at high GTP concentrations. This competition represents a first glimpse at a molecular level explaining how dimerization might regulate the capping process.

Investigation into the Role of the Set2 Auto‐Inhibitory Domain in H3K36 Methyltransferase Activity

Set2 is nucleosomal‐specific methyltransferase that is responsible for mediating histone H3 lysine 36 (H3K36) mono‐, di‐, or trimethylation in Saccharomyces cerevisiae. The human homolog of Set2, SETD2, mediates only H3K36me3. H3K36 methylation has been linked to the prevention of inappropriate or “cryptic” transcription, and SETD2 functions as a tumor repressor through its regulation of the cell cycle and mitotic function. While Set2 catalyzes H3K36 methylation via its SET methyltransferase domain located at its N‐terminus, Set2 contains a Set2‐Rpb1 interaction (SRI) domain at its C‐terminus that binds RNA polymerase II and couples H3K36 methylation with transcriptional elongation. In addition to the SET and SRI domains, a third domain termed the auto inhibitory domain (AID) was identified within Set2 and functions to control the extent of methylation and substrate specificity of Set2's H3K36 methyltransferase activity (1). Additionally, a new study revealed key residues within the AID that are required for the autoinhibitory function of this domain (2). Here, we used an in vitro biochemical approach to further examine the role of the AID in Set2 methyltransferase activity. We designed and expressed full‐length (FL) Set2 containing a recently characterized mutation of the AID (i.e., H336Y) (2) as well as the AID in isolation to test the possibility that this domain directly interacts with the SET domain of Set2 to regulate its H3K36 methylation. Consistent with previous reports, we found that the H336Y AID mutation in Set2 resulted in significantly higher H3K36 methylation activity on nucleosomal and core histone substrates in vitro. Importantly, the normal preference of Set2 for nucleosomes over core histones was lost in the H366Y AID mutation; i.e., the H366Y AID mutation in Set2 enhanced core histone methylation activity to those found with oligonucleosomes. Examination of the AID's ability to interact with Set2's SET domain to regulate its H3K36 methyltransferase activity was next examined. Contrary to our predictions, we found that the addition of AID in Set2 methyltransferase assays did not significantly alter Set2's methyltransferase activity on either oligonucleosomes or core histones. Further preliminary co‐immunoprecipitation analyses did not support a direct interaction of the AID with Set2. These data suggest that the AID's repressive action on Set2 may be regulated through a unique AID structure that exists adjacently to the SET domain which enforces substrate preference and activity rather than by a scenario where the AID interacts directly with, and occludes, the SET domain. Taken together, our data extends our understanding of the autoinhibition functions found in chromatin regulators that direct the precise transcriptional control between chromatin accessibility and repression.Support or Funding InformationNIH to BDSThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metagenomes of a Freshwater Charavirus from British Columbia Provide a Window into Ancient Lineages of Viruses.

Charophyte algae, not chlorophyte algae, are the ancestors of ‘higher plants’; hence, viruses infecting charophytes may be related to those that first infected higher plants. Streamwaters from British Columbia, Canada, yielded single-stranded RNA metagenomes of Charavirus canadensis (CV-Can), that are similar in genomic architecture, length (9593 nt), nucleotide identity (63.4%), and encoded amino-acid sequence identity (53.0%) to those of Charavirus australis (CV-Aus). The sequences of their RNA-dependent RNA-polymerases (RdRp) resemble those found in benyviruses, their helicases those of hepaciviruses and hepegiviruses, and their coat-proteins (CP) those of tobamoviruses; all from the alphavirus/flavivirus branch of the ‘global RNA virome’. The 5’-terminus of the CV-Can genome, but not that of CV-Aus, is complete and encodes a methyltransferase domain. Comparisons of CP sequences suggests that Canadian and Australian charaviruses diverged 29–46 million years ago (mya); whereas, the CPs of charaviruses and tobamoviruses last shared a common ancestor 212 mya, and the RdRps of charaviruses and benyviruses 396 mya. CV-Can is sporadically abundant in low-nutrient freshwater rivers in British Columbia, where Chara braunii, a close relative of C. australis, occurs, and which may be its natural host. Charaviruses, like their hosts, are ancient and widely distributed, and thus provide a window to the viromes of early eukaryotes and, even, Archaea.

The Crg1 N-Terminus Is Essential for Methyltransferase Activity and Cantharidin Resistance in Saccharomyces cerevisiae.

Crg1 is an S-adenosylmethionine (SAM)-dependent methyltransferase required for cantharidin resistance in yeast. Crg1 has a well-characterized methyltransferase domain that inactivates cantharidin by methylation. However, the remaining part of the Crg1 protein is yet to be functionally characterized. In this study, we identified an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance. Yeast cells lacking 41 residues of the N-terminus of Crg1 ( crg1ΔN) showed hypersensitivity to cantharidin as same as the null mutant, crg1. The mass spectrometry-based biochemical enzyme assay revealed a loss of methyltransferase activity in Crg1ΔN, which justifies the loss of cantharidin resistance, as well. The subcellular distribution of Crg1ΔN-daGFP showed cytoplasmic aggregates, whereas wild-type Crg1-daGFP was distributed normally in the cytoplasm. Interestingly, the Crg1-methyltransferase domain point mutants (D44A, D67A, and E105A/D108A) also showed the same cytoplasmic aggregates as Crg1ΔN-daGFP. In silico prediction of the tertiary structures of these mutants indicated an altered protein conformation. Altogether, these observations suggest that the N-terminal truncation, as well as the point mutations in the methyltransferase domain, alters the native folding of Crg1 methyltransferase, resulting in a loss of enzyme activity. Furthermore, the crg1ΔN mutant showed the same phenotypes as the crg1 null mutant in the presence of cantharidin, i.e., lethal cell growth, PE auxotrophy, temperature sensitivity, endoplasmic reticulum stress, GPI anchor missorting, and cell wall damage. Overall, this study identifies an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance.

Characterization and detection of maize-associated pteridovirus (MaPV), infecting maize (Zea mays) in the Arusha region of Tanzania

Maize lethal necrosis disease (MLND) is currently threatening maize production in a large area of East Africa. Synergistic infections of Maize chlorotic mottle virus (MCMV) and members of the Potyviridae family are known to elicit MLND. Metaviromics studies of plant viruses have allowed for the discovery of novel viral diversity, with a number of these viruses having recently been shown to co-occur with MCMV and Potyviruses. However, their contribution to the expression of disease symptoms still requires extensive research. In a survey for viruses associated with maize in Tanzania, 35 samples were sequenced using an RNA-tag-seq metaviromics approach. Bioinformatic analysis of assembled reads yielded two contigs (5.8 and 2.7 kbp) that shared sequence homology with RNA1 and RNA2 of the dsRNA Japanese holly fern mottle virus (JHFMoV) (genus: Pteridovirus) from a single sample collected in the Arusha region. RNA1 encodes for a polyprotein product containing putative viral methyltransferase, helicase and polymerase domains. RNA2 contains three open reading frames (ORFs), one of which encodes for a putative movement protein, while the remaining two encode for putative products with no known function. The tentative name Maize-associated pteridovirus (MaPV) has been proposed for the virus in this study.

Site-specific manipulation of Arabidopsis loci using CRISPR-Cas9 SunTag systems

Understanding genomic functions requires site-specific manipulation of loci via efficient protein effector targeting systems. However, few approaches for targeted manipulation of the epigenome are available in plants. Here, we adapt the dCas9-SunTag system to engineer targeted gene activation and DNA methylation in Arabidopsis. We demonstrate that a dCas9-SunTag system utilizing the transcriptional activator VP64 drives robust and specific activation of several loci, including protein coding genes and transposable elements, in diverse chromatin contexts. In addition, we present a CRISPR-based methylation targeting system for plants, utilizing a SunTag system with the catalytic domain of the Nicotiana tabacum DRM methyltransferase, which efficiently targets DNA methylation to specific loci, including the FWA promoter, triggering a developmental phenotype, and the SUPERMAN promoter. These SunTag systems represent valuable tools for the site-specific manipulation of plant epigenomes.

The ezh2(sa1199) mutant zebrafish display no distinct phenotype.

Polycomb group (PcG) proteins are essential regulators of epigenetic gene silencing and development. The PcG protein enhancer of zeste homolog 2 (Ezh2) is a key component of the Polycomb Repressive Complex 2 and is responsible for placing the histone H3 lysine 27 trimethylation (H3K27me3) repressive mark on the genome through its methyltransferase domain. Ezh2 is highly conserved in vertebrates. We studied the role of ezh2 during development of zebrafish with the use of a mutant allele (ezh2(sa1199), R18STOP), which has a stop mutation in the second exon of the ezh2 gene. Two versions of the same line were used during this study. The first and original version of zygotic ezh2(sa1199) mutants unexpectedly retained ezh2 expression in brain, gut, branchial arches, and eyes at 3 days post-fertilization (dpf), as revealed by in-situ hybridization. Moreover, the expression pattern in homozygous mutants was identical to that of wild types, indicating that mutant ezh2 mRNA is not subject to nonsense mediated decay (NMD) as predicted. Both wild type and ezh2 mutant embryos presented edemas at 2 and 3 dpf. The line was renewed by selective breeding to counter select the non-specific phenotypes and survival was assessed. In contrast to earlier studies on ezh2 mutant zebrafish, ezh2(sa1199) mutants survived until adulthood. Interestingly, the ezh2 mRNA and Ezh2 protein were present during adulthood (70 dpf) in both wild type and ezh2(sa1199) mutant zebrafish. We conclude that the ezh2(sa1199) allele does not exhibit an ezh2 loss-of-function phenotype.

Comparative Analysis of NS5 Protein for Tick Borne Encephalitis Virus Strains in three Virus Subtypes

Non-structural protein 5 (NS5) of tick-borne encephalitis virus is an enzyme which is responsible for a copying of viral RNA, and it has a strong structural similarity to RNA polymerases of another RNA virus families. The strains of the virus are separated into three subtypes, which differ by specific mutations in virus proteins, including NS5 protein. The methods of structural bioinformatics allow to construct a model of NS5 protein for several strains of the virus.The paper presents the comparative analysis of sequences and structures of NS5 protein, for three subtypes of the tick-borne encephalitis virus. The segments of protein were identified where the highest difference between subtypes and within subtypes is observed. These segments, where most of the mutations are accumulated, are located in methyltransferase domain, in the inter-domain interface, and in the three subdomains of polymerase domain. The association between the locations of mutations in NS5 protein and the flexibility of a protein backbone was observed using normal mode analysis. Namely, the most important mutations are located in the parts of protein where the amplitude of synchronous oscillations estimated using normal mode analysis is the highest: in the second zinc binding pocket within polymerase domain, in the N-terminal extension within inter-domain interface, and around an active site of methyltransferase domain.

M58 - FUNCTIONAL ANALYSIS OF THE SCHIZOPHRENIA ASSOCIATED GENE AS3MT IN SH-SY5Y NEUROBLASTOMA CELLS

Background Schizophrenia is a neuropsychiatric disorder with a prevalence of 1%, characterised by episodes of psychosis and an alteration in cognitive function. The aetiology of Schizophrenia is still largely unknown but evidence suggests an underlying neurodevelopmental aspect despite onset occurring in adulthood as well as a considerable genetic burden. Recently 108 genomic loci have robustly been associated with Schizophrenia. However, of these identified genes, very few have been characterised for their role in brain development. Arsenite Methyltransferase (AS3MT) is located in the10q24.32 GWAS locus, which is the most statistically significant locus outside the Major Histocompatibility Complex (MHC) region associated with schizophrenia. Furthermore, both mQTLs and expression quantitative trait loci (eQTL) map to this gene and a recent publication has identified an alternative splice variant which is increased in schizophrenia brain. AS3MT encodes for a methyltransferase involved in arsenic metabolism, however the role of AS3MT in brain development has not been explored. Methods CRISPR-Cas9 technology was used to create a knockout cell line of AS3MT in SH-SY5Y neuroblastoma cells. Two guide RNAs (gRNA) were designed, one to cut in exon 4 and one in exon 6 of the AS3MT gene, where the methyltransferase domain is located. These guides were cloned into two expression vectors which express the gRNA, Cas9 enzyme and either EGFP or mCherry. These vectors were transfected into P11 SH-SY5Y (neuroblastoma) cell line and screened for double fluorescence and sorted into single cells by Fluorescent Activated Cell Sorting after 24 hours. These cells were allowed to clonally expand for 3 weeks before DNA was extracted for genotyping by PCR. Protein expression of AS3MT was measured by western blotting, gene expression by RT-PCR and DNA methylation by the Ilumina EPIC array. Immunocytochemistry techniques will be used to identify any alterations in cell morphology. Results Of the cells transfected with EGPF and mCherry expressing CRISPR-Cas9 vectors 15.5% of the single sorted cells expressed both EGPF and mCherry and were sorted into 96 well plates. Following a week of clonal expansion approximately 38% of wells contained colonies, where 41% un-transfected control sorted cells had colonies. Following genotyping 27.8% (5/18) were identified as heterozygous mutants, 16.7% (3/18) were homozygous mutants and 55.6% (10/18) were wild type. Discussion We have developed a robust protocol for creating deletion mutants using CRISPR-Cas9 technology and a functional domain of AS3MT has been deleted in the neuroblastoma cell line SH-SY5Y using CRISPR-Cas9 technology, further work is ongoing to characterise the molecular consequences of this mutation.

Structural basis of 7SK RNA 5'-γ-phosphate methylation and retention by MePCE.

Among RNA 5′-cap structures, γ-phosphate monomethylation is unique to a small subset of noncoding RNAs, 7SK and U6 in humans. 7SK is capped by methylphosphate capping enzyme (MePCE), which has a second non-enzymatic role as a core component of the 7SK RNP that is an essential regulator of RNA transcription. We report 2.0 and 2.1 Å X-ray crystal structures of human MePCE methyltransferase domain bound to S-adenosylhomocysteine (SAH) and uncapped or capped 7SK substrates, respectively. 7SK recognition is achieved by protein contacts to a 5′ hairpin-single-stranded RNA region, explaining MePCE specificity for 7SK and U6. The structures reveal SAH and product RNA in a near-transition state geometry. Surprisingly, binding experiments show that MePCE has higher affinity for capped vs uncapped 7SK, with kinetic data supporting a slow product release model. This work reveals the molecular mechanism of methyl transfer and 7SK retention by MePCE for subsequent assembly of 7SK RNP.

Structural Basis of DNMT1 and DNMT3A-Mediated DNA Methylation.

DNA methylation, one of the major epigenetic mechanisms, plays critical roles in regulating gene expression, genomic stability and cell lineage commitment. The establishment and maintenance of DNA methylation in mammals is achieved by two groups of DNA methyltransferases (DNMTs): DNMT3A and DNMT3B, which are responsible for installing DNA methylation patterns during gametogenesis and early embryogenesis, and DNMT1, which is essential for propagating DNA methylation patterns during replication. Both groups of DNMTs are multi-domain proteins, containing a large N-terminal regulatory region in addition to the C-terminal methyltransferase domain. Recent structure-function investigations of the individual domains or large fragments of DNMT1 and DNMT3A have revealed the molecular basis for their substrate recognition and specificity, intramolecular domain-domain interactions, as well as their crosstalk with other epigenetic mechanisms. These studies highlight a multifaceted regulation for both DNMT1 and DNMT3A/3B, which is essential for the precise establishment and maintenance of lineage-specific DNA methylation patterns in cells. This review summarizes current understanding of the structure and mechanism of DNMT1 and DNMT3A-mediated DNA methylation, with emphasis on the functional cooperation between the methyltransferase and regulatory domains.

Stuffed Methyltransferase Catalyzes the Penultimate Step of Pyochelin Biosynthesis.

Nonribosomal peptide synthetases use tailoring domains to incorporate chemical diversity into the final natural product. A structurally unique set of tailoring domains are found to be stuffed within adenylation domains and have only recently begun to be characterized. PchF is the NRPS termination module in pyochelin biosynthesis and includes a stuffed methyltransferase domain responsible for S-adenosylmethionine (AdoMet)-dependent N-methylation. Recent studies of stuffed methyltransferase domains propose a model in which methylation occurs on amino acids after adenylation and thiolation rather than after condensation to the nascent peptide chain. Herein, we characterize the adenylation and stuffed methyltransferase didomain of PchF through the synthesis and use of substrate analogues, steady-state kinetics, and onium chalcogen effects. We provide evidence that methylation occurs through an SN2 reaction after thiolation, condensation, cyclization, and reduction of the module substrate cysteine and is the penultimate step in pyochelin biosynthesis.

The Structure of the Bifunctional Everninomicin Biosynthetic Enzyme EvdMO1 Suggests Independent Activity of the Fused Methyltransferase-Oxidase Domains.

Members of the orthosomycin family of natural products are decorated polysaccharides with potent antibiotic activity and complex biosynthetic pathways. The defining feature of the orthosomycins is an orthoester linkage between carbohydrate moieties that is necessary for antibiotic activity and is likely formed by a family of conserved oxygenases. Everninomicins are octasaccharide orthosomycins produced by Micromonospora carbonacea that have two orthoester linkages and a methylenedioxy bridge, three features whose formation logically requires oxidative chemistry. Correspondingly, the evd gene cluster encoding everninomicin D encodes two monofunctional nonheme iron, α-ketoglutarate-dependent oxygenases and one bifunctional enzyme with an N-terminal methyltransferase domain and a C-terminal oxygenase domain. To investigate whether the activities of these domains are linked in the bifunctional enzyme EvdMO1, we determined the structure of the N-terminal methyltransferase domain to 1.1 Å and that of the full-length protein to 3.35 Å resolution. Both domains of EvdMO1 adopt the canonical folds of their respective superfamilies and are connected by a short linker. Each domain's active site is oriented such that it faces away from the other domain, and there is no evidence of a channel connecting the two. Our results support EvdMO1 working as a bifunctional enzyme with independent catalytic activities.

Structural and Functional Studies of a gem-Dimethylating Methyltransferase from a trans-Acyltransferase Assembly Line.

The methyl substituents in products of trans-acyltransferase assembly lines are usually incorporated by S-adenosyl-methionine (SAM)-dependent methyltransferase (MT) domains. The gem-dimethyl moieties within the polyketide disorazol are installed through the iterative action of an MT in the third module of its assembly line. The 1.75-Å-resolution crystal structure of this MT helps elucidate how it catalyzes the addition of two methyl groups. Activity assays of point mutants on β-ketoacyl chains linked to an acyl carrier protein and N-acetylcysteamine provide additional insights into the roles of active site residues. The replacement of an alanine with a phenylalanine at an apparent gatekeeping position resulted in more monomethylation than dimethylation. MTs may form an interface with ketoreductases (KRs) and even mediate the docking of trans-acyltransferase assembly line polypeptides through this association.

Sclerotinia minor Endornavirus 1, a Novel Pathogenicity Debilitation-Associated Mycovirus with a Wide Spectrum of Horizontal Transmissibility.

Sclerotinia minor is a phytopathogenic fungus causing sclerotinia blight on many economically important crops. Here, we have characterized the biological and molecular properties of a novel endornavirus, Sclerotinia minor endornavirus 1 (SmEV1), isolated from the hypovirulent strain LC22 of S. minor. The genome of SmEV1 is 12,626 bp long with a single, large open reading frame (ORF), coding for a putative protein of 4020 amino acids. The putative protein contains cysteine-rich region (CRR), viral methyltransferase (MTR), putative DEXDc, viral helicase (Hel), and RNA-dependent RNA polymerase (RdRp) domains. The putative protein and the conserved domains are phylogenetically related to endornaviruses. SmEV1 does not contain a site-specific nick characteristic of most previously described endornaviruses. Hypovirulence and associated traits of strain LC22 and SmEV1 were readily cotransmitted horizontally via hyphal contact to isolates of different vegetative compatibility groups of S. minor. Additionally, SmEV1 in strain LC22 was found capable of being transmitted vertically through sclerotia. Furthermore, mycelium fragments of hypovirulent strain LC22 have a protective activity against attack by S. minor. Taken together, we concluded that SmEV1 is a novel hypovirulence-associated mycovirus with a wide spectrum of transmissibility, and has potential for biological control (virocontrol) of diseases caused by S. minor.

One-Pot Biocombinatorial Synthesis of Herbicidal Thaxtomins

Thaxtomins are a group of phytotoxic diketopiperazines produced by tens of plant pathogenic Streptomyces strains and have received considerable attention as bioherbicide. To synthesize thaxtomin analogue libraries for herbicide development, we here develop an in vitro biocombinatorial approach. We first prepared two recombinant single-module multifunctional nonribosomal peptide synthetases (NRPSs), TxtA and TxtB. Biochemical studies revealed that TxtA and TxtB tolerated small substituents on the aromatic moiety of l-Phe and l-Trp, respectively. Intriguingly, TxtB showed a control of the substrate scopes of TxtA and a previously characterized, pathway-specific nitration promoting P450 TxtE. We further demonstrated that the methyltransferase (MT) domain of TxtA and TxtB posed a minimal influence on the assembly of the diketopiperazine core by other domains of these two enzymes, providing a way for structural diversification. The pathway-specific bifunctional P450 TxtC was then recombinantly produced in Esch...

Purification and functional characterization of tomato mosaic virus 130K protein expressed in silkworm pupae using a baculovirus vector

Tomato mosaic virus (ToMV; genus, Tobamovirus) is a member of the alpha-like virus superfamily of positive-strand RNA viruses, which includes many plant and animal viruses of agronomical and clinical importance. The genomes of alpha-like viruses encode replication-associated proteins that contain methyltransferase, helicase and/or polymerase domains. The three-dimensional structure of the helicase domain fragment of ToMV has been determined, but the structures of the other domains of alpha-like virus replication proteins are not available. In this study, we expressed full-length ToMV replication-associated protein 130 K, which contains the methyltransferase and helicase domains, using the baculovirus-silkworm expression system and purified the recombinant protein to near homogeneity. Purified 130 K, which was stable in phosphate buffer containing magnesium ions and ATP, formed a dimer in solution and hydrolyzed nucleoside 5′-triphosphates.

Strobilurin biosynthesis in Basidiomycete fungi

Strobilurins from fungi are the inspiration for the creation of the β-methoxyacrylate class of agricultural fungicides. However, molecular details of the biosynthesis of strobilurins have remained cryptic. Here we report the sequence of genomes of two fungi that produce strobilurins and show that each contains a biosynthetic gene cluster, which encodes a highly reducing polyketide synthase with very unusual C-terminal hydrolase and methyltransferase domains. Expression of stpks1 in Aspergillus oryzae leads to the production of prestrobilurin A when the fermentation is supplemented with a benzoyl coenzyme A (CoA) analogue. This enables the discovery of a previously unobserved route to benzoyl CoA. Reconstruction of the gene cluster in A. oryzae leads to the formation of prestrobilurin A, and addition of the gene str9 encoding an FAD-dependent oxygenase leads to the key oxidative rearrangement responsible for the creation of the β-methoxyacrylate toxophore. Finally, two methyltransferases are required to complete the synthesis.

Methylation of Structured RNA by the m6A Writer METTL16 Is Essential for Mouse Embryonic Development

SummaryInternal modification of RNAs with N6-methyladenosine (m6A) is a highly conserved means of gene expression control. While the METTL3/METTL14 heterodimer adds this mark on thousands of transcripts in a single-stranded context, the substrate requirements and physiological roles of the second m6A writer METTL16 remain unknown. Here we describe the crystal structure of human METTL16 to reveal a methyltransferase domain furnished with an extra N-terminal module, which together form a deep-cut groove that is essential for RNA binding. When presented with a random pool of RNAs, METTL16 selects for methylation-structured RNAs where the critical adenosine is present in a bulge. Mouse 16-cell embryos lacking Mettl16 display reduced mRNA levels of its methylation target, the SAM synthetase Mat2a. The consequence is massive transcriptome dysregulation in ∼64-cell blastocysts that are unfit for further development. This highlights the role of an m6A RNA methyltransferase in facilitating early development via regulation of SAM availability.