Mechanism of Radical Initiation in the Radical SAM Enzyme Superfamily.

Radical S-adenosylmethionine (SAM) enzymes use a site-differentiated [4Fe-4S] cluster and SAM to initiate radical reactions through liberation of the 5'-deoxyadenosyl (5'-dAdo•) radical. They form the largest enzyme superfamily, with more than 700,000 unique sequences currently, and their numbers continue to grow as a result of ongoing bioinformatics efforts. The range of extremely diverse, highly regio- and stereo-specific reactions known to be catalyzed by radical SAM superfamily members is remarkable. The common mechanism of radical initiation in the radical SAM superfamily is the focus of this review. Most surprising is the presence of an organometallic intermediate, Ω, exhibiting an Fe-C5'-adenosyl bond. Regioselective reductive cleavage of the SAM S-C5' bond produces 5'-dAdo• to form Ω, with the regioselectivity originating in the Jahn-Teller effect. Ω liberates the free 5'-dAdo• as the catalytically active intermediate through homolysis of the Fe-C5' bond, in analogy to Co-C5' bond homolysis in B12, which was once viewed as biology's choice of radical generator.

Macrocyclization and Backbone Modification in RiPP Biosynthesis.

The past decade has seen impressive advances in understanding the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs). One of the most common modifications found in these natural products is macrocyclization, a strategy also used by medicinal chemists to improve metabolic stability and target affinity and specificity. Another tool of the peptide chemist, modification of the amides in a peptide backbone, has also been observed in RiPPs. This review discusses the molecular mechanisms of biosynthesis of a subset of macrocyclic RiPP families, chosen because of the unusual biochemistry involved: the five classes of lanthipeptides (thioether cyclization by Michael-type addition), sactipeptides and ranthipeptides (thioether cyclization by radical chemistry), thiopeptides (cyclization by [4+2] cycloaddition), and streptide (cyclization by radical C-C bond formation). In addition, the mechanisms of backbone amide methylation, backbone epimerization, and backbone thioamide formation are discussed, as well as an unusual route to small molecules by posttranslational modification.

Splice site m6A methylation prevents binding of U2AF35 to inhibit RNA splicing.

The N6-methyladenosine (m6A) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an m6A mark on the 3' splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3' splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3' splice site. We propose that use of splice-site m6A is an ancient mechanism for splicing regulation.

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.

Biosynthesis of the Metalloclusters of Nitrogenases.

Nitrogenase is a versatile metalloenzyme that is capable of catalyzing two important reactions under ambient conditions: the reduction of nitrogen (N2) to ammonia (NH3), a key step in the global nitrogen cycle; and the reduction of carbon monoxide (CO) and carbon dioxide (CO2) to hydrocarbons, two reactions useful for recycling carbon waste into carbon fuel. The molybdenum (Mo)- and vanadium (V)-nitrogenases are two homologous members of this enzyme family. Each of them contains a P-cluster and a cofactor, two high-nuclearity metalloclusters that have crucial roles in catalysis. This review summarizes the progress that has been made in elucidating the biosynthetic mechanisms of the P-cluster and cofactor species of nitrogenase, focusing on what is known about the assembly mechanisms of the two metalloclusters in Mo-nitrogenase and giving a brief account of the possible assembly schemes of their counterparts in V-nitrogenase, which are derived from the homology between the two nitrogenases.

Structural insights into DNMT5-mediated ATP-dependent high-fidelity epigenome maintenance.

Epigenetic evolution occurs over million-year timescales in Cryptococcus neoformans and is mediated by DNMT5, the first maintenance type cytosine methyltransferase identified in the fungal or protist kingdoms, the first dependent on adenosine triphosphate (ATP), and the most hemimethyl-DNA-specific enzyme known. To understand these novel properties, we solved cryo-EM structures of CnDNMT5 in three states. These studies reveal an elaborate allosteric cascade in which hemimethylated DNA binding first activates the SNF2 ATPase domain by a large rigid body rotation while the target cytosine partially flips out of the DNA duplex. ATP binding then triggers striking structural reconfigurations of the methyltransferase catalytic pocket to enable cofactor binding, completion of base flipping, and catalysis. Bound unmethylated DNA does not open the catalytic pocket and is instead ejected upon ATP binding, driving high fidelity. This unprecedented chaperone-like, enzyme-remodeling role of the SNF2 ATPase domain illuminates how energy is used to enable faithful epigenetic memory.

Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence.

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate epigenetic states and subsequent gene expression. This metabolism-epigenome link sensitizes chromatin methylation to altered SAM abundance, yet the mechanisms that allow organisms to adapt and protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear mono-methylation of H3 Lys 9 (H3K9) at the expense of broad losses in histone di- and tri-methylation. Under SAM-depleted conditions, H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide evidence for an adaptive response that enables epigenetic persistence to metabolic stress.

Demethylation of the Protein Phosphatase PP2A Promotes Demethylation of Histones to Enable Their Function as a Methyl Group Sink.

Dysregulation of chromatin methylation is associated with defects in cellular differentiation as well as a variety of cancers. How cells regulate the opposing activities of histone methyltransferase and demethylase enzymes to set the methylation status of the epigenome for proper control of gene expression and metabolism remains poorly understood. Here, we show that loss of methylation of the major phosphatase PP2A in response to methionine starvation activates the demethylation of histones through hyperphosphorylation of specific demethylase enzymes. In parallel, this regulatory mechanism enables cells to preserve SAM by increasing SAH to limit SAM consumption by methyltransferase enzymes. Mutants lacking the PP2A methyltransferase or the effector H3K36 demethylase Rph1 exhibit elevated SAM levels and are dependent on cysteine due to reduced capacity to sink the methyl groups of SAM. Therefore, PP2A directs the methylation status of histones by regulating the phosphorylation status of histone demethylase enzymes in response to SAM levels.

METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation.

7-methylguanosine (m7G) is present at mRNA caps and at defined internal positions within tRNAs and rRNAs. However, its detection within low-abundance mRNAs and microRNAs (miRNAs) has been hampered by a lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in miRNAs. Using this technique (Borohydride Reduction sequencing [BoRed-seq]) alongside RNA immunoprecipitation, we identify m7G within a subset of miRNAs that inhibit cell migration. We show that the METTL1 methyltransferase mediates m7G methylation within miRNAs and that this enzyme regulates cell migration via its catalytic activity. Using refined mass spectrometry methods, we map m7G to a single guanosine within the let-7e-5p miRNA. We show that METTL1-mediated methylation augments let-7 miRNA processing by disrupting an inhibitory secondary structure within the primary miRNA transcript (pri-miRNA). These results identify METTL1-dependent N7-methylation of guanosine as a new RNA modification pathway that regulates miRNA structure, biogenesis, and cell migration.

Essential role of N-terminal SAM regions in STIM1 multimerization and function.

The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.

Identification of two archaeal GDGT lipid-modifying proteins reveals diverse microbes capable of GMGT biosynthesis and modification.

Archaea produce unique membrane-spanning lipids (MSLs), termed glycerol dialkyl glycerol tetraethers (GDGTs), which aid in adaptive responses to various environmental challenges. GDGTs can be modified through cyclization, cross-linking, methylation, hydroxylation, and desaturation, resulting in structurally distinct GDGT lipids. Here, we report the identification of radical SAM proteins responsible for two of these modifications-a glycerol monoalkyl glycerol tetraether (GMGT) synthase (Gms), responsible for covalently cross-linking the two hydrocarbon tails of a GDGT to produce GMGTs, and a GMGT methylase (Gmm), capable of methylating the core hydrocarbon tail. Heterologous expression of Gms proteins from various archaea in Thermococcus kodakarensis results in the production of GMGTs in two isomeric forms. Further, coexpression of Gms and Gmm produces mono- and dimethylated GMGTs and minor amounts of trimethylated GMGTs with only trace GDGT methylation. Phylogenetic analyses reveal the presence of Gms homologs in diverse archaeal genomes spanning all four archaeal superphyla and in multiple bacterial phyla with the genetic potential to synthesize fatty acid-based MSLs, demonstrating that GMGT production may be more widespread than previously appreciated. We demonstrate GMGT production in three Gms-encoding archaea, identifying an increase in GMGTs in response to elevated temperature in two Archaeoglobus species and the production of GMGTs with up to six rings in Vulcanisaeta distributa. The occurrence of such highly cyclized GMGTs has been limited to environmental samples and their detection in culture demonstrates the utility of combining genetic, bioinformatic, and lipid analyses to identify producers of distinct archaeal membrane lipids.

Local auxin biosynthesis promotes shoot patterning and stem cell differentiation in Arabidopsis shoot apex.

The shoot apical meristem (SAM) of higher plants comprises distinct functional zones. The central zone (CZ) is located at the meristem summit and harbors pluripotent stem cells. Stem cells undergo cell division within the CZ and give rise to descendants, which enter the peripheral zone (PZ) and become recruited into lateral organs. Stem cell daughters that are pushed underneath the CZ form rib meristem (RM). To unravel the mechanism of meristem development, it is essential to know how stem cells adopt distinct cell fates in the SAM. Here, we show that meristem patterning and floral organ primordia formation, besides auxin transport, are regulated by auxin biosynthesis mediated by two closely related genes of the TRYPTOPHAN AMINOTRANSFERASE family. In Arabidopsis SAM, TAA1 and TAR2 played a role in maintaining auxin responses and the identity of PZ cell types. In the absence of auxin biosynthesis and transport, the expression pattern of the marker genes linked to the patterning of the SAM is perturbed. Our results prove that local auxin biosynthesis, in concert with transport, controls the patterning of the SAM into the CZ, PZ and RM.

S-adenosyl-L-methionine supplementation alleviates damaged intestinal epithelium and inflammatory infiltration caused by Mat2a deficiency.

Methionine is important for intestinal development and homeostasis in various organisms. However, the underlying mechanisms are poorly understood. Here, we demonstrate that the methionine adenosyltransferase gene Mat2a is essential for intestinal development and that the metabolite S-adenosyl-L-methionine (SAM) plays an important role in intestinal homeostasis. Intestinal epithelial cell (IEC)-specific knockout of Mat2a exhibits impaired intestinal development and neonatal lethality. Mat2a deletion in the adult intestine reduces cell proliferation and triggers IEC apoptosis, leading to severe intestinal epithelial atrophy and intestinal inflammation. Mechanistically, we reveal that SAM maintains the integrity of differentiated epithelium and protects IECs from apoptosis by suppressing the expression of caspases 3 and 8 and their activation. SAM supplementation improves the defective intestinal epithelium and reduces inflammatory infiltration sequentially. In conclusion, our study demonstrates that methionine metabolism and its intermediate metabolite SAM play essential roles in intestinal development and homeostasis in mice.

CryoSegNet: accurate cryo-EM protein particle picking by integrating the foundational AI image segmentation model and attention-gated U-Net.

Picking protein particles in cryo-electron microscopy (cryo-EM) micrographs is a crucial step in the cryo-EM-based structure determination. However, existing methods trained on a limited amount of cryo-EM data still cannot accurately pick protein particles from noisy cryo-EM images. The general foundational artificial intelligence-based image segmentation model such as Meta's Segment Anything Model (SAM) cannot segment protein particles well because their training data do not include cryo-EM images. Here, we present a novel approach (CryoSegNet) of integrating an attention-gated U-shape network (U-Net) specially designed and trained for cryo-EM particle picking and the SAM. The U-Net is first trained on a large cryo-EM image dataset and then used to generate input from original cryo-EM images for SAM to make particle pickings. CryoSegNet shows both high precision and recall in segmenting protein particles from cryo-EM micrographs, irrespective of protein type, shape and size. On several independent datasets of various protein types, CryoSegNet outperforms two top machine learning particle pickers crYOLO and Topaz as well as SAM itself. The average resolution of density maps reconstructed from the particles picked by CryoSegNet is 3.33 Å, 7% better than 3.58 Å of Topaz and 14% better than 3.87 Å of crYOLO. It is publicly available at https://github.com/jianlin-cheng/CryoSegNet.

Structural Basis for Regulation of METTL16, an S-Adenosylmethionine Homeostasis Factor.

S-adenosylmethionine (SAM) is an essential metabolite that acts as a cofactor for most methylation events in the cell. The N6-methyladenosine (m6A) methyltransferase METTL16 controls SAM homeostasis by regulating the abundance of SAM synthetase MAT2A mRNA in response to changing intracellular SAM levels. Here we present crystal structures of METTL16 in complex with MAT2A RNA hairpins to uncover critical molecular mechanisms underlying the regulated activity of METTL16. The METTL16-RNA complex structures reveal atomic details of RNA substrates that drive productive methylation by METTL16. In addition, we identify a polypeptide loop in METTL16 near the SAM binding site with an autoregulatory role. We show that mutations that enhance or repress METTL16 activity in vitro correlate with changes in MAT2A mRNA levels in cells. Thus, we demonstrate the structural basis for the specific activity of METTL16 and further suggest the molecular mechanisms by which METTL16 efficiency is tuned to regulate SAM homeostasis.

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

Internal 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.

Facile and dynamic cleavage of every iron-sulfide bond in cuboidal iron-sulfur clusters.

Nature employs weak-field metalloclusters to support a wide range of biological processes. The most ubiquitous metalloclusters are the cuboidal Fe-S clusters, which are comprised of Fe sites with locally high-spin electronic configurations. Such configurations enhance rates of ligand exchange and imbue the clusters with a degree of structural plasticity that is increasingly thought to be functionally relevant. Here, we examine this phenomenon using isotope tracing experiments. Specifically, we demonstrate that synthetic [Fe4S4] and [MoFe3S4] clusters exchange their Fe atoms with Fe2+ ions dissolved in solution, a process that involves the reversible cleavage and reformation of every Fe-S bond in the cluster core. This exchange is facile-in most cases occurring at room temperature on the timescale of minutes-and documented over a range of cluster core oxidation states and terminal ligation patterns. In addition to suggesting a highly dynamic picture of cluster structure, these results provide a method for isotopically labeling pre-formed clusters with spin-active nuclei, such as 57Fe. Such a protocol is demonstrated for the radical S-adenosyl-l-methionine enzyme, RlmN.

SAM68 is required for regulation of Pumilio by the NORAD long noncoding RNA.

The number of known long noncoding RNA (lncRNA) functions is rapidly growing, but how those functions are encoded in their sequence and structure remains poorly understood. NORAD (noncoding RNA activated by DNA damage) is a recently characterized, abundant, and highly conserved lncRNA that is required for proper mitotic divisions in human cells. NORAD acts in the cytoplasm and antagonizes repressors from the Pumilio family that bind at least 17 sites spread through 12 repetitive units in NORAD sequence. Here we study conserved sequences in NORAD repeats, identify additional interacting partners, and characterize the interaction between NORAD and the RNA-binding protein SAM68 (KHDRBS1), which is required for NORAD function in antagonizing Pumilio. These interactions provide a paradigm for how repeated elements in a lncRNA facilitate function.

From powerhouse to processing plant: conserved roles of mitochondrial outer membrane proteins in tRNA splicing.

The mitochondrial cytoplasmic surface serves as a processing site for numerous RNAs from budding yeast to metazoans. We report that budding yeast mitochondrial outer membrane (MOM) proteins that are subunits of the translocase of the outer mitochondrial membrane (Tom70 and Tom 22) and sorting and assembly machinery (Sam37) are required for efficient pretransfer RNA (pre-tRNA) splicing. Defective pre-tRNA splicing in MOM mutants is due not to loss of respiratory metabolism but instead inefficient targeting/tethering of tRNA splicing endonuclease (SEN) subunits to mitochondria. Schizosaccharomyces pombe SEN subunits also localize to mitochondria, and Tom70 is required for this localization and pre-tRNA splicing. Thus, the role of MOM protein in targeting/tethering SEN subunits to mitochondria has been conserved for >500 million years.

Structural analysis of the SAM domain of the Arabidopsis mitochondrial tRNA import receptor.

Mitochondria are membrane-bound organelles of endosymbiotic origin with limited protein-coding capacity. The import of nuclear-encoded proteins and nucleic acids is required and essential for maintaining organelle mass, number, and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAalavia conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with six monomers per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of the Tric1 SAM domain may play a role in protein function whereby mutation of Gly241 introducing a larger side chain at this position disrupted the oligomer and resulted in the loss of RNA binding capability. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype. AlphaFold2 structure prediction of both the SAM domain and Tric1 support a cyclic pentameric or hexameric structure. In the case of a hexameric structure, a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane is observed. Our results highlight the importance of oligomerization of Tric1 for protein function.