Fig. 3: Mapping Ψ sites in human mt-tRNAs.
From: Complete chemical structures of human mitochondrial tRNAs
a A CID spectrum of the RNase T1-digested fragment of human mt-tRNACys. The doubly charged negative ion of the cyanoethylated RNA fragment (m/z 1345.7) was used as the precursor for CID. Asterisks in the spectrum denote product ions containing ce1Ψ. b A CID spectrum of the cyanoethylated RNA fragment to determine the site for Ψs in human mt-tRNAPro. The triply charged negative ion of the mono-cyanoethylated RNA fragment (m/z1266.5) was used as the precursor for CID. Assignment of the product ions revealed the presence of two fragments containing ce1Ψ66 and ce1Ψ67. Asterisks in the spectrum denote product ions containing ce1Ψ. c CMC-PE analysis for detection of Ψ sites in human mt-tRNACys. HeLa total RNA treated with (+) or without (−) CMC was reverse-transcribed with a primer specific to human mt-tRNACys. The sequence ladders for U and A were generated under the same conditions in the presence of ddATP and ddTTP, respectively. The positions for Ψs and sequence are shown with the gel image. Source data are provided as a Source Data file. d Diagram of tRNA-Ψ-seq. Detailed procedure is described in the “Methods” section. e IGV snapshots of tRNA-Ψ-seq for human mt-tRNAGly and mt-tRNAVal. Histograms of mapped reads are shown for each tRNA when tRNA-Ψ-seq was performed in the absence (−) or presence (+) of CMC. The RT-stop signature for CMC-Ψ (indicated by red arrowheads) can be observed as a sudden drop in the number of piled reads.
