Journal: Nucleic Acids Research

Article Title: Investigation of TRMT61B methyltransferase activity on mRNA and its effects on translation

doi: 10.1093/nar/gkag365

Figure Lengend Snippet: Overexpressed TRMT61B methylates thousands of sites across the transcriptome. ( A ) Schematic of TRMT61B overexpression (OE) in U-2 OS cells and m 1 A-IP. RNA was purified, polyA-selected, immunoprecipitated, and then prepared into libraries with a TruSeq Stranded mRNA Library Prep kit (Illumina). Both control and TRMT61B OE m 1 A-IP were carried out in duplicate. ( B ) Volcano plot showing DESeq2 results depicting fold changes in transcript abundance in m 1 A-IP samples compared to input samples under TRMT61B OE conditions. Top 30 hits by -log 10 (p adj ) are labeled, and mitochondrial genes are highlighted in red. ( C ) Volcano plot showing single-nucleotide sites called by bakR as having altered misincorporation levels in TRMT61B OE conditions versus control OE conditions, with “high-confidence” YMRA sites (bakR hits with additional filtering of P < .05, difference in log-odds of mutation rate >1, >1% mutation rate in both replicates, >5% average mutation rate in IP, and <5% average mutation rate in input samples) highlighted in red. Top 30 hits are labeled. ( D ) RNA types of all high-confidence single-nucleotide sites, regardless of methylated motif. ( E ) Regions of modification for all high-confidence sites, regardless of methylated motif. ( F ) Top motif discovered in high-confidence sites using STREME , covering 74.3% of all sites. ( G ) RNA type of high-confidence sites, filtered to only YMRA-motif sites. ( H ) Regions of modification for high-confidence sites, filtered to only YMRA-motif sites. ( I ) Metagene plot showing distribution of YMRA-containing high-confidence sites.

Article Snippet: U-2 OS cells (HTB-96) were obtained from the American Tissue Culture Collection and maintained in McCoy’s 5A Medium (Gibco, 16600082) with 10% fetal bovine serum and 1× penicillin–streptomycin at 37°C under a humidified atmosphere with 5% CO 2 .

Techniques: Over Expression, Purification, Immunoprecipitation, Control, Labeling, Mutagenesis, Methylation, Modification

Journal: Environmental and Molecular Mutagenesis

Article Title: Oxidative DNA Damage Exacerbates the Mutagenic Potential of Alternative DNA Structures via Altered DNA Repair Processing

doi: 10.1002/em.70059

Figure Lengend Snippet: Mutation frequencies of B‐DNA and H‐DNA sequences with or without oxidative damage and XPA, a key NER protein. B‐DNA‐ or H‐DNA‐forming reporter sequences were exposed to different levels of oxidative stress (−, +, or ++) and then transfected into human U2OS cell lines with either (A and B) wild type or (C and D) XPA knockout phenotypes to allow for DNA repair processing. Mutation frequencies were quantified (A and C) using a blue‐white screening assay. Each condition represents the average of at least three replicates (+SEM). Statistical comparisons between conditions were performed using a two‐way ANOVA with significance indicated as p < 0.05 (*), or < 0.01 (**). Mutation spectra were then determined (B and D) using Sanger sequencing, with at least 15 mutants analyzed per condition and classified as point mutations (1 bp), small deletions (< 15 bp), or large deletions (> 15 bp). Mutation spectra ratios are shown per condition as a ratio of mutation frequency.

Article Snippet: Wild‐type U2OS cells (ATCC, #HTB‐96) were used unless otherwise specified, including the CRISPR‐Cas9 knockout U2OS cell lines for either OGG1, APE1, or XPA (provided by Dr. Robert Sobol, Brown University) and previously characterized (Bordelon ).

Techniques: Mutagenesis, Transfection, Knock-Out, Screening Assay, Sequencing

Journal: Environmental and Molecular Mutagenesis

Article Title: Oxidative DNA Damage Exacerbates the Mutagenic Potential of Alternative DNA Structures via Altered DNA Repair Processing

doi: 10.1002/em.70059

Figure Lengend Snippet: Mutation frequencies of B‐DNA and H‐DNA sequences with or without oxidative damage and key BER proteins. B‐DNA‐ or H‐DNA‐forming reporter sequences were exposed to increasing levels of oxidative stress (−, +, or ++) and then transfected into human U2OS cell lines with either (A and B) OGG1 knockout or (C and D) APE1 knockout phenotypes to allow for DNA repair processing. Mutation frequencies were then quantified (A and C) using a blue‐white screening assay. Each condition represents the average of at least three replicates (+SEM). Statistical comparisons between conditions were performed using a two‐way ANOVA with significance indicated as p < 0.05 (*), < 0.01 (**), or < 0.001 (***). Mutation spectra were then determined (B and D) using Sanger sequencing, with at least 15 mutants analyzed per condition and classified as point mutations (1 bp), small deletions (< 15 bp), or large deletions (> 15 bp). Mutation spectra ratios are shown per condition as a ratio of mutation frequency.

Article Snippet: Wild‐type U2OS cells (ATCC, #HTB‐96) were used unless otherwise specified, including the CRISPR‐Cas9 knockout U2OS cell lines for either OGG1, APE1, or XPA (provided by Dr. Robert Sobol, Brown University) and previously characterized (Bordelon ).

Techniques: Mutagenesis, Transfection, Knock-Out, Screening Assay, Sequencing

Journal: Environmental and Molecular Mutagenesis

Article Title: Oxidative DNA Damage Exacerbates the Mutagenic Potential of Alternative DNA Structures via Altered DNA Repair Processing

doi: 10.1002/em.70059

Figure Lengend Snippet: Association of XPA and APE1 proteins with B‐DNA or H‐DNA sequences with or without oxidative damage and key BER or NER proteins. B‐DNA‐ or H‐DNA‐forming reporter sequences were first exposed to increasing levels of oxidative stress (−, +, or ++). Reporter sequences were then transfected into human U2OS cell lines with (A) wild‐type (previously published matched control (Zewail‐Foote et al. )), (B) XPA knockout, or (C) APE1 knockout phenotypes for DNA repair processing. Chromatin immunoprecipitation (ChIP) was then used to measure the association of XPA or APE1 proteins with B‐DNA or H‐DNA sequences. Protein association is shown as a percentage of total input DNA (% input). Each condition shows the average % input of three replicates (+SEM). Condition comparisons use a Wilcoxon rank sums approach demonstrating p < 0.05 (*), or < 0.01 (**).

Article Snippet: Wild‐type U2OS cells (ATCC, #HTB‐96) were used unless otherwise specified, including the CRISPR‐Cas9 knockout U2OS cell lines for either OGG1, APE1, or XPA (provided by Dr. Robert Sobol, Brown University) and previously characterized (Bordelon ).

Techniques: Transfection, Control, Knock-Out, Chromatin Immunoprecipitation