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

Journal: Nucleic Acids Research

Article Title: OT-Curtains: an approach for studying protein interactions with DNA ends using optical tweezers and confocal fluorescence microscopy

doi: 10.1093/nar/gkag370

Figure Lengend Snippet: OT-Curtains substrate schematics. ( a ) Plasmid containing four poly-BbvCI regions. ( b ) Site-specific nicking by Nt.BbvCI creates five nicks per poly-BbvCI region. ( c ) OT-Curtains backbone molecule with four 63-nt ssDNA gaps. ( d – f ) The OT-Curtains construct is assembled by incorporating biotinylated handles and branch structures of choice to the backbone. Variants addressed in this work have either long branches (d) or short branches (e), but different structural elements can be incorporated (f), depending on the biological question to be addressed.

Article Snippet: The 22 142 bp 4x poly-BbvCI plasmid (see previous section) at a final concentration of 2.7 nM is digested with 1.25 units of BssHII (New England Biolabs, #R0199S) to linearize the DNA in 6.25 μl buffer provided by the manufacturer.

Techniques: Plasmid Preparation, Construct