Search Results (44)

DNA methylation is a conserved and vital epigenetic modification that plays essential roles in plant growth, development, and responses to environmental stress. Cytosine-5 DNA methyltransferases (C5-MTases) and DNA demethylases (dMTases) are key regulators of DNA methylation dynamics. However, a comprehensive characterization of these gene families in sweet potato has remained elusive. In this study, we systematically identified and analyzed eight C5-MTase and five dMTase genes in the genomes of diploid (Ipomoea trifida, 2n = 2x = 30) and autohexaploid (Ipomoea batatas, 2n = 6x = 90) sweet potato. Phylogenetic, structural, and synteny analyses revealed a high degree of conservation among these genes, suggesting their essential roles during evolution. Promoter analysis uncovered multiple cis-acting elements, particularly those responsive to light and hormones. In addition, we examined the expression profiling of IbC5-MTases and IbdMTases genes during storage root development, revealing that several were highly expressed during the early and rapid expansion stages. These findings suggest that C5-MTases and dMTases may contribute to the regulation of storage root formation in sweet potato through epigenetic mechanisms, offering valuable insights for future functional studies and epigenetic breeding efforts. Full article

Ribose 2′-O-methylation (Nm), a key RNA modification, is catalyzed by diverse 2′-O-methyltransferases (2′-O-MTases), yet the evolutionary trajectories of these enzymes remain poorly studied. Here, with a comprehensive collection of functionally validated 2′-O-MTases, we classified them into 11 families based on the distinct methyltransferase (MTase) domains. Homology searches across 198 species identified 6746 proteins, revealing the widespread distribution of 2′-O-MTases across the Tree of Life. Eight MTase domains (e.g., FtsJ, SpoU-methylase) existed both in eukaryotes and prokaryotes, indicating their ancient origin in the Last Universal Common Ancestor (LUCA). In contrast, the AdoMet-MTase, TRM13, and Trm56 domains are lineage-specific. Copy number expansion of most 2′-O-MTase families occurred as life evolved from prokaryotes to eukaryotes, where they might engage in more complex regulation of cell differentiation and development. Domain composition, Ka/Ks ratio, and domain structural analyses showed that purifying selection conserved catalytic domains across most families, despite the frequent integration of auxiliary domains. Notably, the FtsJ family diverged into three deeply separated lineages via remodeling the catalytic pocket, with each lineage specializing in the methylation of mRNA caps, rRNA, or tRNA. These findings illuminate the evolutionary trajectory of 2′-O-MTases, highlighting their ancient multiple origins and functional diversification. Full article

The design and synthesis of antiviral compounds were guided by computationally predicted data against highly conserved non-structural proteins (Nsps) of the SARS-CoV-2 coronavirus. Chromenephenylmethanone-1 (CPM-1), a novel biphenylpyran (BPP), was selected from a unique natural product library based on in silico docking scores to coronavirus Nsps with high specificity to the methyltransferase protein (2′-O-MTase, Nsp10–16), which is responsible for viral mRNA maturation and host innate immune response evasion. To target the 2′-O-MTase, CPM-1, along with intermediate BPP regioisomers, tetrahydrophenylmethanones (TPMs), were synthesized and structurally validated via nuclear magnetic resonance (NMR) data and DP4+ structure probability analyses. To investigate the activity of these BPPs, the following in vitro assays were conducted: SARS-CoV-2 inhibition, biochemical target validation, mutagenicity, and cytotoxicity. CPM-1 possessed notable activity against SARS-CoV-2 with 98.9% inhibition at 10 µM and an EC₅₀ of 7.65 µM, as well as inhibition of SARS-CoV-2’s 2′-O-MTase (expressed and purified) with an IC₅₀ of 1.5 ± 0.2 µM. In addition, CPM-1 revealed no cytotoxicity (CC₅₀ of >100 µM) or mutagenicity (no frameshift or base-pair mutations). This study demonstrates the potential of computational modeling for the discovery of natural product prototypes followed by the design and synthesis of drug leads to inhibit the SARS-CoV-2 2′-O-MTase. Full article

The dual methyltransferase methyltransferase-like protein 13, also referred to as METTL13, or formerly known as FEAT (faintly expressed in healthy tissues, aberrantly overexpressed in tumors), has garnered attention as a significant enzyme in various cancer types, as evidenced by prior literature reviews. Recent studies have shed light on new potential roles for METTL13, hinting at its promise as a therapeutic target. This review aims to delve into the multifaceted biology of METTL13, elucidating its proposed mechanisms of action, regulatory pathways, and its implications in disease states, as supported by the current body of literature. Furthermore, the review will highlight emerging trends and gaps in our understanding of METTL13, paving the way for future research efforts. By contextualizing METTL13 within the broader landscape of cancer biology and therapeutics, this study serves as an introductory guide to METTL13, aiming to provide readers with a thorough understanding of its role in disease phenotypes. Full article

The ongoing COVID-19 pandemic still threatens human health around the world. The methyltransferases (MTases) of SARS-CoV-2, specifically nsp14 and nsp16, play crucial roles in the methylation of the N7 and 2′-O positions of viral RNA, making them promising targets for the development of antiviral drugs. In this work, we performed structure-based virtual screening for nsp14 and nsp16 using the screening workflow (HTVS, SP, XP) of Schrödinger 2019 software, and we carried out biochemical assays and molecular dynamics simulation for the identification of potential MTase inhibitors. For nsp14, we screened 239,000 molecules, leading to the identification of three hits A1–A3 showing N7-MTase inhibition rates greater than 60% under a concentration of 50 µM. For the SAM binding and nsp10-16 interface sites of nsp16, the screening of 210,000 and 237,000 molecules, respectively, from ZINC15 led to the discovery of three hit compounds B1–B3 exhibiting more than 45% of 2′-O-MTase inhibition under 50 µM. These six compounds with moderate MTase inhibitory activities could be used as novel candidates for the further development of anti-SARS-CoV-2 drugs. Full article

Zika virus (ZIKV) is a positive-sense single-stranded virus member of the Flaviviridae family. Among other arboviruses, ZIKV can cause neurological disorders such as Guillain Barré syndrome, and it can have congenital neurological manifestations and affect fertility. ZIKV nonstructural protein 5 (NS5) is essential for viral replication and limiting host immune detection. Herein, we performed virtual screening to identify novel small-molecule inhibitors of the ZIKV NS5 methyltransferase (MTase) domain. Compounds were tested against the MTases of both ZIKV and DENV, demonstrating good inhibitory activities against ZIKV MTase. Extensive molecular dynamic studies conducted on the series led us to identify other derivatives with improved activity against the MTase and limiting ZIKV infection with an increased selectivity index. Preliminary pharmacokinetic parameters have been determined, revealing excellent stability over time. Preliminary in vivo toxicity studies demonstrated that the hit compound 17 is well tolerated after acute administration. Our results provide the basis for further optimization studies on novel non-nucleoside MTase inhibitors. Full article

Clostridium carboxidivorans P7 (DSM 15243) is a bacterium that converts syngas (a mixture of CO, H₂, and CO₂) into hexanol. An optimized and scaled-up industrial process could therefore provide a renewable source of fuels and chemicals while consuming industry waste gases. However, the genetic engineering of this bacterium is hindered by its multiple restriction–modification (RM) systems: the genome of C. carboxidivorans encodes at least ten restriction enzymes and eight methyltransferases (MTases). To gain insight into the complex RM systems of C. carboxidivorans, we analyzed genomic methylation patterns using single-molecule real-time (SMRT) sequencing and bisulfite sequencing. We identified six methylated sequence motifs. To match the methylation sites to the predicted MTases of C. carboxidivorans, we expressed them individually in Escherichia coli for functional characterization. Recognition motifs were identified for all three Type I MTases (CAYNNNNNCTGC/GCAGNNNNNRTG, CCANNNNNNNNTCG/CGANNNNNNNNTGG and GCANNNNNNNTNNCG/CGNNANNNNNNNTGC), two Type II MTases (GATAAT and CRAAAAR), and a single Type III MTase (GAAAT). However, no methylated recognition motif was found for one of the three Type II enzymes. One recognition motif that was methylated in C. carboxidivorans but not in E. coli (AGAAGC) was matched to the remaining Type III MTase through a process of elimination. Understanding these enzymes and the corresponding recognition sites will facilitate the development of genetic tools for C. carboxidivorans that can accelerate the industrial exploitation of this strain. Full article

When compared with bacteria, relatively little is known about the restriction–modification (RM) systems of archaea, particularly those in taxa outside of the haloarchaea. To improve our understanding of archaeal RM systems, we surveyed REBASE, the restriction enzyme database, to catalog what is known about the genes and activities present in the 519 completely sequenced archaeal genomes currently deposited there. For 49 (9.4%) of these genomes, we also have methylome data from Single-Molecule Real-Time (SMRT) sequencing that reveal the target recognition sites of the active m⁶A and m⁴C DNA methyltransferases (MTases). The gene-finding pipeline employed by REBASE is trained primarily on bacterial examples and so will look for similar genes in archaea. Nonetheless, the organizational structure and protein sequence of RM systems from archaea are highly similar to those of bacteria, with both groups acquiring systems from a shared genetic pool through horizontal gene transfer. As in bacteria, we observe numerous examples of “persistent” DNA MTases conserved within archaeal taxa at different levels. We experimentally validated two homologous members of one of the largest “persistent” MTase groups, revealing that methylation of C(m⁵C)WGG sites may play a key epigenetic role in Crenarchaea. Throughout the archaea, genes encoding m⁶A, m⁴C, and m⁵C DNA MTases, respectively, occur in approximately the ratio 4:2:1. Full article

DNA methylation plays an indispensable role in genome stability, regulation of gene expression and plant stress response. It is mediated by DNA methyltransferases (MTases). Twelve putative MTases of P. betulaefolia were identified and were classified into MET1, CMT, DRM2 and Dnmt2 groups based on the organization of various characteristic domains. Three pairs of paralogous genes were identified with the Ka/Ks ratio varied from 0.232 for PbeMET1a and PbeMET1b to 0.251 for PbeCMT2 and PbeCMT3, respectively. In addition, the Ka/Ks ratio for nine pairs of orthologous gene pairs between P. betulaefolia and apple were varied from 0.053 for PbeDRM3 and MD17G1031900 to 0.278 for PbeDnmt2b and MD15G1120500, respectively. All the 12 members of MTase genes were located on nine chromosomes out of 17 P. betulaefolia chromosomes with highly conserved protein sequence structures. The isoelectric points (pI) of MTases ranged from 4.74 to 7.24, while molecular weight varied from 35.99 to 174.32. The expression profiles of MTase and other salt-responsive genes under salt stress treatment revealed their important roles involved in salt response in P. betulaefolia. Furthermore, three selected salt-responsive genes (PbeNHX2.1, PbeCBL2 and PbeAKT2) were found altered in methylation level of promoters (which contain CpG islands) under salt stress. Especially, the PbeAKT2 promoter regions showed high CHG and CHH methylation types. Our study provided a genome-wide survey of the MTase gene family and highlighted their roles in salt response. These results also provided an effective way for the breeding and improvement of salt-tolerant pear varieties. Full article

Zika virus (ZIKV) is a type of RNA virus that belongs to the Flaviviridae family. We have reported the construction of a DNA-launched replicon of the Asian-lineage Natal RGN strain and the production of single-round infectious particles (SRIPs) via the combination of prM/E virus-like particles with the replicon. The main objective of the study was to engineer the ZIKV replicon as mammalian expression vectors and evaluate the potential of ZIKV mini-replicon-based SRIPs as delivery vehicles for heterologous gene expression in vitro and in vivo. The mini-replicons contained various genetic elements, including NS4B, an NS5 methyltransferase (MTase) domain, and an NS5 RNA-dependent RNA polymerase (RdRp) domain. Among these mini-replicons, only ZIKV mini-replicons 2 and 3, which contained the full NS5 and NS4B-NS5 genetic elements, respectively, exhibited the expression of reporters (green fluorescent protein (GFP) and cyan fluorescent protein–yellow fluorescent fusion protein (CYP)) and generated self-replicating RNAs. When the mini-replicons were transfected into the cells expressing ZIKV prM/E, this led to the production of ZIKV mini-replicon-based SRIPs. ZIKV mini-replicon 3 SRIPs showed a significantly higher yield titer and a greater abundance of self-replicating replicon RNAs when compared to ZIKV mini-replicon 2 SRIPs. Additionally, there were disparities in the dynamics of CYP expression and cytotoxic effects observed in various infected cell types between ZIKV mini-replicon 2-CYP and 3-CYP SRIPs. In particular, ZIKV mini-replicon 3-CYP SRIPs led to a substantial decrease in the survival rates of infected cells at a MOI of 2. An in vivo gene expression assay indicated that hACE2 expression was detected in the lung and brain tissues of mice following the intravenous administration of ZIKV mini-replicon 3-hACE2 SRIPs. Overall, this study highlights the potential of ZIKV mini-replicon-based SRIPs as promising vehicles for gene delivery applications in vitro and in vivo. Full article

Bacterial virulence, persistence and defence are affected by epigenetic modifications, including DNA methylation. Solitary DNA methyltransferases modulate a variety of cellular processes and influence bacterial virulence; as part of a restriction-modification (RM) system, they act as a primitive immune system in methylating the own DNA, while unmethylated foreign DNA is restricted. We identified a large family of type II DNA methyltransferases in Metamycoplasma hominis, comprising six solitary methyltransferases and four RM systems. Motif-specific 5mC and 6mA methylations were identified with a tailored Tombo analysis on Nanopore reads. Selected motifs with methylation scores >0.5 fit with the gene presence of DAM1 and DAM2, DCM2, DCM3, and DCM6, but not for DCM1, whose activity was strain-dependent. The activity of DCM1 for C^mCWGG and of both DAM1 and DAM2 for G^mATC was proven in methylation-sensitive restriction and finally for recombinant rDCM1 and rDAM2 against a dam-, dcm-negative background. A hitherto unknown dcm8/dam3 gene fusion containing a (TA) repeat region of varying length was characterized within a single strain, suggesting the expression of DCM8/DAM3 phase variants. The combination of genetic, bioinformatics, and enzymatic approaches enabled the detection of a huge family of type II DNA MTases in M. hominis, whose involvement in virulence and defence can now be characterized in future work. Full article

The human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus. It is the major cause of severe acute lower respiratory tract infection in infants, the elderly population, and immunocompromised individuals. There is still no approved vaccine or antiviral treatment against RSV disease, but new monoclonal prophylactic antibodies are yet to be commercialized, and clinical trials are in progress. Hence, urgent efforts are needed to develop efficient therapeutic treatments. RSV RNA synthesis comprises viral transcription and replication that are catalyzed by the large protein (L) in coordination with the phosphoprotein polymerase cofactor (P), the nucleoprotein (N), and the M2-1 transcription factor. The replication/transcription is orchestrated by the L protein, which contains three conserved enzymatic domains: the RNA-dependent RNA polymerase (RdRp), the polyribonucleotidyl transferase (PRNTase or capping), and the methyltransferase (MTase) domain. These activities are essential for the RSV replicative cycle and are thus considered as attractive targets for the development of therapeutic agents. In this review, we summarize recent findings about RSV L domains structure that highlight how the enzymatic activities of RSV L domains are interconnected, discuss the most relevant and recent antivirals developments that target the replication/transcription complex, and conclude with a perspective on identified knowledge gaps that enable new research directions. Full article

Since late 2016, a yellow fever virus (YFV) variant carrying a set of nine amino acid variations has circulated in South America. Three of them were mapped on the methyltransferase (MTase) domain of viral NS5 protein. To assess whether these changes affected viral infectivity, we synthesized YFV carrying the MTase of circulating lineage as well as its isoform with the residues of the previous strains (NS5 K101R, NS5 V138I, and NS5 G173S). We observed a slight difference in viral growth properties and plaque phenotype between the two synthetic YFVs. However, the MTase polymorphisms associated with the Brazilian strain of YFV (2016–2019) confer more susceptibility to the IFN-I. In addition, in vitro MTase assay revealed that the interaction between the YFV MTase and the methyl donor molecule (SAM) is altered in the Brazilian MTase variant. Altogether, the results reported here describe that the MTase carrying the molecular signature of the Brazilian YFV circulating since 2016 might display a slight decrease in its catalytic activity but virtually no effect on viral fitness in the parameters comprised in this study. The most marked influence of these residues stands in the immune escape against the antiviral response mediated by IFN-I. Full article

Methyltransferases (MTases) enzymes, responsible for RNA capping into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are emerging important targets for the design of new anti-SARS-CoV-2 agents. Here, analogs of S-adenosylmethionine (SAM), obtained from the bioisosteric substitution of the sulfonium and amino acid groups, were evaluated by rigorous computational modeling techniques such as molecular dynamics (MD) simulations followed by relative binding free analysis against nsp16/nsp10 complex from SARS-CoV-2. The most potent inhibitor (2a) shows the lowest binding free energy (–58.75 Kcal/mol) and more potency than Sinefungin (SFG) (–39.8 Kcal/mol), a pan-MTase inhibitor, which agrees with experimental observations. Besides, our results suggest that the total binding free energy of each evaluated SAM analog is driven by van der Waals interactions which can explain their poor cell permeability, as observed in experimental essays. Overall, we provide a structural and energetic analysis for the inhibition of the nsp16/nsp10 complex involving the evaluated SAM analogs as potential inhibitors. Full article

In this work, a novel ratiometric electrochemical readout platform was proposed and developed for the fast and flexible analysis of M.SssI methyltransferase (MTase) activity. In this platform, two hairpin DNAs (H1 and H2) were designed. H1 contains the palindromic sequence of 5′-CCGG-3′ in its stem which could be methylated and hybridize with H2 labeled by methylene blue (MB) as one of the signal reporters on a gold electrode (GE) in the presence of M.SssI MTase. Additionally, a specific immunoreaction was introduced by conjugating an anti-5-methylcytosine antibody, a DNA CpG methylation recognition unit, with 1,3-ferrocenedicarboxylic acid (Fc) as the second signal reporter. The results showed that when the Fc tag approaches, the MB tag was far from the gold electrode surface, resulting in a decrease in the oxidation peak current of MB (I_MB) and an increase in the oxidation peak current of Fc (I_Fc). The ratiometric electrochemical method above shows the linear range of detection was 0 U/mL 40 U/mL with a detection limit of 0.083 U/mL (the mean signal of blank measures þ3s). Full article