Journal: Nucleic Acids Research

Article Title: Structural basis for sensitivity and acquired resistance of fungal cap guanine-N7 methyltransferases to the antifungal antibiotic sinefungin

doi: 10.1093/nar/gkaf538

Figure Lengend Snippet: Structures of K. lactis RNA cap methyltransferase in complexes with products SAH and m 7 GTP. ( A ) Schmatic outline of the experimental setup for methylation assays. Methylation of substrate (m 7 GpppA) was detected by separating reactions products (in red and blue) using a high-performance liquid chromatography (HPLC) column coupled to a triple quadrupole (QQQ) MS, which detects product ions (see the “Materials and methods” section). Quantification was achieved by normalizing ion counts to an internal standard, l -tryptophan. The ratio of m 7 GpppA ion counts to l -tryptophan ion counts yielded the internal standard ratio (ISTD). A range of m 7 GpppA calibration standards (with constant l -tryptophan) provided a linear calibration curve between ISTD and m 7 GpppA concentration. Sample ISTDs were then used to interpolate a discrete m 7 GpppA concentration from the calibration curve. ( B ) Methylation activities of wild-type (purple circles) and two truncated Kl Abd1 variants ( Kl Abd1Δ116, pink circles; and Kl Abd1Δ137, dark pink circles) with GpppA substrate (see the “Materials and methods” section). Error bars (one standard deviation) are calculated from three independent experiments performed in triplicate. ( C ) A view of the Kl Abd1 structure (colored purple) as a ribbon with arrows for β-strands and wide ribbons for helices. A transparent molecular surface envelops the structure. N and C denote amino and carboxyl termini, respectively. Bound SAH in the methyl donor site is shown in stick representation (green). ( D ) A closeup view of the methyl donor site of Kl Abd1–SAH complex. ( E ) A view of the methyl acceptor site of Kl Abd1 bound to m 7 GTP (stick representation in cyan). Bound adenine in the methyl donor site is shown in stick representation (green) and rest of the unresolved substrate/product is depicted in thin gray line. Side chains shown in stick representation, and waters are denoted by red spheres. Atomic contacts are indicated by dashed lines with distances. ( F ) Aligned primary structures of cap methyltransferases from K. lactis ( Kl Abd1), S. cerevisiae ( Sc Abd1), E. cuniculi (Ecm1), and Homo sapiens (RNMT). The secondary structure elements of Kl Abd1 are shown above the amino acid sequences, with α-helices depicted as cylinders and β-strand as arrows. Gaps in the alignments are indicated by “•”. Side-chain identity/similarity is denoted by shading and letter color (purple shade conserved in all; purple letters conserved in most). A predicted disordered region N-terminal to the catalytic domain is denoted by a purple dashed line above the alignment. Kl Abd1 amino acids that contact SAH and m 7 GTP are indicated by green and orange circles, respectively. A conserved tyrosine in β11 that interacts with the cap guanosine is outlined in cyan.

Article Snippet: Solutions of SAM (Cayman Chemical), SAH (Millipore-Sigma), SFG (Santa Cruz Biotechnology), GTP (Jena Bioscience), m 7 GTP (Millipore-Sigma), GpppA (Jena Bioscience), and m 7 GpppA (Jena Bioscience) were prepared in 300 mM HEPES buffer (pH 7.5), and concentrations were determined from the absorbance at 260 nm using the following extinction coefficients (M −1 cm −1 ): SAM, 16000; SAH, 15400; SFG, 16000; GTP, 13700; m 7 GTP, 9800; GpppA, 27000; and m 7 GpppA, 22700.

Techniques: Methylation, High Performance Liquid Chromatography, Targeted Proteomics, Concentration Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Structural basis for sensitivity and acquired resistance of fungal cap guanine-N7 methyltransferases to the antifungal antibiotic sinefungin

doi: 10.1093/nar/gkaf538

Figure Lengend Snippet: Activity and inhibition of S. cerevisiae Abd1. ( A ) Methylation activities of full-length wild-type (WT) Sc Abd1 (light gray triangles), two truncated Sc Abd1 variants ( Sc Abd1Δ119, gray circles; and Sc Abd1Δ140, dark gray squares) and SFG-resistant Sc Abd1-E124K-K163R-K311R-F387Y-Y416F variant (red squares) with GpppA substrate (see the “Materials and methods” section). Error bars (one standard deviation) are calculated from three independent experiments performed in triplicate. ( B ) Inhibition of methylation activities (three independent experiments) of Sc Abd1WT (light gray circles), SFG-resistant variants of Sc Abd1 ( Sc Abd1-L59P-Y416C, green circles; Sc Abd1-E124K-K163-K311R-F387Y-Y416, red circles; Sc Abd1-Y416N-R422H, black circles; Sc Abd1-Y416A, orange circles) with SAH. Reaction mixtures containing 250 nM enzyme preparations were incubated with 100 μM SAM, 250 μM GpppA, and variable concentrations (0, 3.125, 12.5, 25, 50, 100, 200, and 800 μM; see the “Materials and methods” section) of SAH at 30°C in buffer consisting of 50 mM Tris (pH 8.2), 200 mM NaCl, and 5 mM βME. After 20 min, 10 μl reaction samples were quenched with 10 μl of quenching solution containing 100 mM H 2 SO 4 and 50 μM l -tryptophan. Concurrently, m 7 GpppA standards were quenched in quenching solution. ( C ) Inhibition of methylation activities of wild-type and SFG-resistant variants of Sc Abd1 with SFG as shown in panel (B). Reaction mixture containing 250 nM enzyme preparations was incubated with 100 μM SAM, 250 μM GpppA, and variable concentrations (0, 15.6, 31.25, 62.5, 125, 250, and 500 nM for the wild-type proteins; 0, 100 nM, 500 nM, 750 nM, 1.2 μM, 1.7 μM, 2.2 μM, and 4.0 μM for the variants; see the “Materials and methods” section) of SFG at 30°C in buffer consisting of 50 mM Tris (pH 8.2), 200 mM NaCl, and 5 mM βME. After 20 min, 10 μl reaction samples were quenched with 10 μl of quenching solution containing 100 mM H 2 SO 4 and 50 μM l -tryptophan. ( D ) Ratios of IC 50 values of wild-type and variant Sc Abd1 and Kl Abd1 between SAH and SFG with GpppA substrate. Significance between the wild-type and resistant variants determined by ordinary one-way ANOVA (**** signifies adjusted P -value <.0001). ( E ) IC 50 values for SAH (μM) and SFG (nM) obtained from panels (B) and (C) for Sc Abd1WT (gray), Sc Abd1-L59P-Y416C (green), Sc Abd1-E124K-K163-K311R-F387Y-Y416 (red), Sc Abd1-Y416N-R422H (black), and Sc Abd1-Y416A (orange).

Article Snippet: Solutions of SAM (Cayman Chemical), SAH (Millipore-Sigma), SFG (Santa Cruz Biotechnology), GTP (Jena Bioscience), m 7 GTP (Millipore-Sigma), GpppA (Jena Bioscience), and m 7 GpppA (Jena Bioscience) were prepared in 300 mM HEPES buffer (pH 7.5), and concentrations were determined from the absorbance at 260 nm using the following extinction coefficients (M −1 cm −1 ): SAM, 16000; SAH, 15400; SFG, 16000; GTP, 13700; m 7 GTP, 9800; GpppA, 27000; and m 7 GpppA, 22700.

Techniques: Activity Assay, Inhibition, Methylation, Variant Assay, Standard Deviation, Incubation

Journal: Nucleic Acids Research

Article Title: Structural basis for sensitivity and acquired resistance of fungal cap guanine-N7 methyltransferases to the antifungal antibiotic sinefungin

doi: 10.1093/nar/gkaf538

Figure Lengend Snippet: Structure of K. lactis Abd1 bound to inhibitor SFG and SFG plus GTP. ( A ) SAH inhibition. The extent of methylation of GpppA by wild-type Kl Abd1 was quantified (see the “Materials and methods” section) in the presence of increasing concentrations (0, 3.125, 12.5, 25, 50, 100, 200, and 800 μM) of SAH and the obtained relative activities (%) for the enzyme (green circles) were plotted as a function SAH concentrations to determine the IC 50 value (indicated). ( B ) SFG inhibition. The extent of methylation of GpppA by wild-type Kl Abd1 was quantified (see the “Materials and methods” section) in the presence of increasing concentrations (0, 15.6, 32.25, 62.5, 125, 250, and 500 nM) of SFG and the relative activities (%) for Kl Abd1 (light yellow circles) were plotted as a function SFG concentrations to determine the IC 50 value (indicated). Error bars (one standard deviation) are calculated from three independent experiments performed in triplicate. ( C ) A view of the methyl donor site of Kl Abd1 bound to SFG shown in stick representation and colored light yellow. ( D ) A closeup view of the methyl acceptor site of Kl Abd1 bound to SFG (as in panel B) and GTP (stick representation in cyan). Side chains are shown in stick representation (as in Fig. ), and waters are denoted by red spheres. Atomic contacts are indicated by dashed lines with distances.

Article Snippet: Solutions of SAM (Cayman Chemical), SAH (Millipore-Sigma), SFG (Santa Cruz Biotechnology), GTP (Jena Bioscience), m 7 GTP (Millipore-Sigma), GpppA (Jena Bioscience), and m 7 GpppA (Jena Bioscience) were prepared in 300 mM HEPES buffer (pH 7.5), and concentrations were determined from the absorbance at 260 nm using the following extinction coefficients (M −1 cm −1 ): SAM, 16000; SAH, 15400; SFG, 16000; GTP, 13700; m 7 GTP, 9800; GpppA, 27000; and m 7 GpppA, 22700.

Techniques: Inhibition, Methylation, Standard Deviation

Journal: PLoS ONE

Article Title: The Detergent-Soluble Cytoplasmic Pool of Survivin Suppresses Anoikis and Its Expression Is Associated with Metastatic Disease of Human Colon Cancer

doi: 10.1371/journal.pone.0055710

Figure Lengend Snippet: The experimental protocol was illustrated in Figure S1. Transfection frequencies were checked by using fluorescence microscopy and confirmed to be 80–90%. Cells were kept in serum-free medium for 24–72 h, harvested, and lysed in Laemmli SDS-sample buffer for immunoblot analysis with anti-β-actin, anti-Bax, anti-Smac/DIABLO, anti-XIAP, anti-IκB-α, anti-NF-κB, anti-JNK, anti-c-Jun-P(S73), anti-c-Jun, anti-FAK-P(Y397), and anti-FAK.

Article Snippet: Anti-GFP antibody (JL-8, Clontech Laboratories), anti-Survivin antibody (NB 500-201, Novus Biologicals; NB 500-237, Novus Biologicals; sc-10811, Santa Cruz Biotechnology), anti-activated caspase-3 antibody (#9661, Cell Signaling Technology), anti-LC3B antibody (#4445, Cell Signaling Technology), anti-α-tubulin antibody (CLT9002, Cedarlane Laboratories), anti-β-actin antibody (A1978, Sigma-Aldrich), anti-Bax antibody (sc-493, Santa Cruz Biotechnology), anti-Smac/DIABLO antibody (PM004, MBL International), anti-XIAP antibody (AF822, R&D Systems), anti-IκB-α antibody (sc-371, Santa Cruz Biotechnology), anti-NF-κB antibody (sc-372, Santa Cruz Biotechnology), anti-JNK antibody (sc-474, Santa Cruz Biotechnology), anti-phosphorylated c-Jun at serine 73 antibody (#06-659, EMD Millipore), anti-c-Jun antibody (#09-754, EMD Millipore), anti-phosphorylated FAK at tyrosine 397 antibody (F25420, BD Biosciences), and anti-FAK antibody (sc-932, Santa Cruz Biotechnology) were used for Immunoblot analysis.

Techniques: Transfection, Fluorescence, Microscopy, Western Blot

Journal: Advanced Science

Article Title: The m7G Methyltransferase Mettl1 Drives Cardiac Hypertrophy by Regulating SRSF9‐Mediated Splicing of NFATc4

doi: 10.1002/advs.202308769

Figure Lengend Snippet: YY1 transcriptionally activates Mettl1 in hypertrophic mouse hearts. A) Western blotting analyses and quantification of Mettl1 protein levels in human heart tissues from heart failure patients and non‐heart failure patients (n = 3). B) Dot blot analysis of m7G modification levels in 10‐week TAC heart tissue, with methylene blue (MB) staining as control (n = 3). C) qRT‐PCR analysis of Mettl1 mRNA levels in the control group or the TAC group (n = 4–6). D) Western blot analysis of Mettl1 protein levels in 10‐week TAC heart tissue, with GAPDH as a control (n = 8‐9). E) Representative immunofluorescence images of α‐actinin‐ and Mettl1‐staining in the myocardial sections from the TAC‐ and sham‐operated groups, Scale bar: 50 µ m (n = 3 mice). F) qRT‐PCR analysis of Mettl1 mRNA levels in heart tissue 4 weeks after Ang II infusion (n = 4‐5). G) Western blot analysis of Mettl1 protein levels in heart tissue 4 weeks after Ang II infusion (n = 5). H) qRT‐PCR was conducted to detect Mettl1 mRNA expression in NMCMs infected with YY1‐siRNA or YY1‐siNC followed by stimulation with Ang II (1 µ m ) for 48 h (n = 3). I) Protein levels of Mettl1 in NMCMs infected with YY1‐siRNA or YY1‐siNC followed by stimulation with Ang II (1 µ m ) for 48 h (n = 3). J) NMCMs were infected with YY1‐siRNA or YY1‐siNC, an empty vector, or Mettl1 promoter followed by treatment with Ang II (1 µ m ) for 48 h. Luciferase activity was determined (n = 6). K) ChIP‐qPCR analysis was performed with YY1 or IgG antibody to determine the binding ability of YY1 to Mettl1 promoter in NMCMs after incubating with Ang II (1 µ m ) for 48 h (n = 3).

Article Snippet: Briefly, RIP was performed using m7G (#RN017M, MBL, Tokyo, Japan, 10 µg per sample) and SRSF9 (RN081PW, MBL, Tokyo, Japan, 10 µg per sample) or negative control immunoglobulin G (IgG; using Millipore, USA) of the same species according to the manufacturer's instructions.

Techniques: Western Blot, Dot Blot, Modification, Staining, Control, Quantitative RT-PCR, Immunofluorescence, Expressing, Infection, Plasmid Preparation, Luciferase, Activity Assay, Binding Assay

Journal: Advanced Science

Article Title: The m7G Methyltransferase Mettl1 Drives Cardiac Hypertrophy by Regulating SRSF9‐Mediated Splicing of NFATc4

doi: 10.1002/advs.202308769

Figure Lengend Snippet: SRSF9 is a target of Mettl1‐mediated m7G modification. A) A cross‐tabulation analysis of MeRIP‐seq showing the intersections of upregulated genes (AAV9‐Null vs AAV9‐Mettl1, FC > 1.5) following enhanced m7G modifications in NMCMs (Adv‐Null vs Adv‐Mettl1, FC > 3) and myocardium (AAV9‐Null vs AAV9‐Mettl1, FC > 1.5). B) The Vann plot shows the 19 potential target genes of Mettl1 obtained from screening based on combined KEGG and GO analyses. C) Effects of Mettl1 on the m7G modification of CREB1, SETDB2, PTGS2, and SRSF9 assessed by RNA‐binding protein immunoprecipitation (RIP) in NMCM transfected with Adv‐null or Adv‐Mettl1 (n = 3). D) Visualization of the m7G methylation site on the SRSF9 gene. E) The decay rate of SRSF9 analyzed by qRT‐PCR in NMCMs treated with 5 µg mL −1 actinomycin D for 0, 2, 4, and 6 h after transfection with siNC or siMettl1 for 48 h (n = 7–9). F) The decay rate of SRSF9 determined by qRT‐PCR in NMCMs treated with 5 µg mL −1 actinomycin D for 0, 2, 4, and 6 h after transfection with Adv‐Null or Adv‐Mettl1 for 48 h (n = 8–9). G) Western blot analysis of SRSF9 protein levels in heart tissues from WT or Mettl1 +/− mice of the sham and TAC groups (n = 7). H) Western blot analysis of SRSF9 protein levels in cardiac tissues from mice treated with AAV9‐Null or AAV9‐Mettl1 for 8 weeks (n = 6). I) Western blot analysis of SRSF9 protein levels in NMCMs transfected with siNC or siMettl1 and pretreated with Ang II for 48 h (n = 7). J) Western blot analysis of SRSF9 protein levels in NMCMs treated with Adv‐Null or Adv‐Mettl1 for 48 h (n = 6). K) A graphical illustration of the construction of the plasmid carrying SRSF9 mutation sites (G‐to‐T mutation) and the specific primer was designed for MeRIP‐qPCR and RNA decay analysis. L) SRSF9 wild‐type (SRSF9‐WT, CDS+3′UTR) or SRSF9‐mutant (SRSF9‐Mut, CDS+3′UTR‐mutant) plasmids were co‐transfected with Adv‐Mettl1 into NMCMs, respectively. The effect of Mettl1 on m7G modification of exogenous SRSF9 transcript was assessed by RIP‐qPCR with specific primer as indicated in Figure (n = 4). M) The decay rates of exogenous SRSF9 transcript analyzed by qRT‐PCR in NMCM treated with 5 µg mL −1 actinomycin D for 0, 2, 4, and 6 h after transfection with Adv‐Mettl1 for 48 h (n = 6).

Article Snippet: Briefly, RIP was performed using m7G (#RN017M, MBL, Tokyo, Japan, 10 µg per sample) and SRSF9 (RN081PW, MBL, Tokyo, Japan, 10 µg per sample) or negative control immunoglobulin G (IgG; using Millipore, USA) of the same species according to the manufacturer's instructions.

Techniques: Modification, RNA Binding Assay, Immunoprecipitation, Transfection, Methylation, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Mutagenesis

Journal: Advanced Science

Article Title: The m7G Methyltransferase Mettl1 Drives Cardiac Hypertrophy by Regulating SRSF9‐Mediated Splicing of NFATc4

doi: 10.1002/advs.202308769

Figure Lengend Snippet: M7G methyltransferase targets the SRSF9/NFATc4 axis to regulate cardiac hypertrophy. Transcription factor YY1 is responsible for the upregulation of Mettl1 in cardiac hypertrophy. Upregulated Mettl1 methylates SRSF9 mRNA to increase its stability in an m7G‐dependent manner, which in turn promotes intron retention‐type splicing and stabilization of NFATc4, leading to cardiac hypertrophic growth. (Created with BioRender.com).

Article Snippet: Briefly, RIP was performed using m7G (#RN017M, MBL, Tokyo, Japan, 10 µg per sample) and SRSF9 (RN081PW, MBL, Tokyo, Japan, 10 µg per sample) or negative control immunoglobulin G (IgG; using Millipore, USA) of the same species according to the manufacturer's instructions.

Techniques: