Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Inhibition of tRNA charging activates the ISR without inducing RNP granules. ( A ) U-2 OS cells were treated with 0.2% DMSO carrier, 20 µM halofuginone (HF), 250 µM sodium arsenite (As), or 1.25 µM thapsigargin (Tg) for 1 h, and Western blotting for phosphorylated and total eIF2α was done. Total protein ( n = 3 independent replicates) and molecular weights (in kilodaltons) are shown. ( B ) Translation activity was measured using bioorthogonal noncanonical amino acid tagging in cells treated as in A . Methionine or azidohomoalanine (AHA) was added 10 min prior to collection. The ratio of nascent to total protein is shown from n = 3 independent replicates. ( C ) U-2 OS cells stably expressing GFP-G3BP1 (green) were treated as in A and fixed, and nuclei were stained with Hoechst (blue). The percentage of cells with SGs was quantified from n = 3 independent experiments; ≥305 cells were counted in each condition. Scale bars, 10 µm. ( D ) Cells were treated as in C , and immunofluorescence and fluorescence in situ hybridization were done to detect UBAP2L (magenta), PABPC1 (cyan), and poly(A) RNA with oligo(dT) probes (yellow) ( n = 2 independent experiments). ( E ) Cells were treated with 0.2% DMSO, 20 µM HF plus 0.2% DMSO, 250 µM arsenite, or 1.25 µM thapsigargin for 1 h, and the percentage of cells with SGs was quantified ( n = 3 independent experiments); ≥287 cells were counted per treatment. ( F ) Cells were treated, and immunofluorescence microscopy was done as in D to detect the P-body marker DCP1A. Quantification of the percentage of cells with P-bodies (PBs) from n = 3 independent experiments is shown; ≥364 cells were counted per treatment. ( G ) Cells were treated as in C to detect XRN1 (magenta) and EDC4 (yellow) by immunofluorescence microscopy ( n = 2 independent experiments). ( H ) U-2 OS cells were treated as in E , and immunofluorescence microscopy was done to detect DCP1A. The percentage of cells with PBs from n = 3 independent experiments is shown; ≥351 cells were counted per treatment across all replicates. Representative images are shown with the average ± SEM for each experiment, and green, gray, and pink points represent the average of each replicate. Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005, (****) P < 0.001.

Article Snippet: PABPC1 , Proteintech , 66809 , 1:100 (immunofluorescence).

Techniques: Inhibition, Western Blot, Activity Assay, Stable Transfection, Expressing, Staining, Immunofluorescence, Fluorescence, In Situ Hybridization, Microscopy, Marker

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: PolyA-RNA selection was then carried out using the NEBNext ® High-Input Poly(A) mRNA Isolation Module (NEB, E3370S), following manufacturer protocols.

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

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: Purified TRMT61B methylates hundreds of sites in a synthetic human mRNA 5′UTR/CDS pool. ( A ) Schematic of in vitro TRMT61B methylation of RNA, with validation by LC–MS/MS and detection of single-nucleotide sites using uMRT-based misincorporation. ( B ) SDS–PAGE gel showing purified TRMT61B protein, indicated by the arrow (~57 kDa). ( C ) LC–MS/MS measurements of d 3 m 1 A in a short oligo based on 16S rRNA (39 nucleotides, centered around adenosine) methylated by purified TRMT61B. ( D ) LC–MS/MS measurements of d 3 m 1 A in a 5′UTR/CDS pool methylated by purified TRMT61B. Individual values and mean from three technical replicates is shown for (C) and (D). ( E ) Single-nucleotide sites detected in both high- (HE) and low-enzyme (LE) 5′UTR/CDS pool samples. “High-confidence” YMRA-containing sites (1% mutation rate in two of three replicates in both HE and LE treatments, p adj < .05 in HE sample) are highlighted in red, with top 20 hits labeled. ( F ) Top motif surrounding high-confidence sites (with duplicated sequences from alternative isoforms removed) using STREME , covering 61.5% of all sites. ( G ) Codon positions of high-confidence YMRA single-nucleotide sites located in the CDS. ( H ) Distribution of the number of high-confidence 5′UTR/CDS pool YMRA m 1 A sites found in codons of all possible amino acids. Amino acids with codons that can accommodate a YMRA motif are bolded. ( I ) Metagene plot showing distribution of YMRA-containing high-confidence sites across 5′UTR and CDS regions.

Article Snippet: PolyA-RNA selection was then carried out using the NEBNext ® High-Input Poly(A) mRNA Isolation Module (NEB, E3370S), following manufacturer protocols.

Techniques: Purification, In Vitro, Methylation, Biomarker Discovery, Liquid Chromatography with Mass Spectroscopy, SDS Page, Mutagenesis, Labeling

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: Randomization of methylated sequences confirms upstream YMR sequence preference for TRMT61B. ( A ) Table of the seven mRNA examples chosen for randomized motif experiments, including context sequence and location of single-nucleotide site. ( B ) Schematic of randomized motif experiments, showing randomization of upstream and downstream 3-mer followed by methylation and targeted uMRT-PCR. ( C ) Example data from SEC62 and EZHIP sequences showing nucleotide abundances at each position between −3 and +3 surrounding the modification site of interest. Amplicon sequencing results were split into “unmodified” samples that had no misincorporation at the modification site, while “modified” sequences contained a misincorporation. Results for the other five transcripts are shown in C. Minimum free-energy (MFE) secondary structures surrounding seven example m 1 A sites, generated using RNAfold . The upstream YMR motif is highlighted in yellow, and m 1 A site in red. ( E ) Sequences and MFE secondary structures of five high-confidence m 1 A sites identified in TRMT61B mRNA under conditions of TRMT61B overexpression. The average mutation rates from TRMT61B OE input and IP samples are also included.

Article Snippet: PolyA-RNA selection was then carried out using the NEBNext ® High-Input Poly(A) mRNA Isolation Module (NEB, E3370S), following manufacturer protocols.

Techniques: Methylation, Sequencing, Modification, Amplification, Generated, Over Expression, Mutagenesis