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Overexpressed TRMT61B methylates thousands of sites across the transcriptome. ( A ) Schematic of TRMT61B overexpression (OE) <t>in</t> <t>U-2</t> 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.
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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.

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