Direct Sequencing of tRNA by 2D-HELS-AA MS Seq Reveals Its Different Isoforms and Dynamic Base Modifications

作者：Ning Zhang, Shundi Shi, Xuanting Wang, Wenhao Ni, Xiaohong Yuan, Jiachen Duan, Tony Z. Jia, Barney Yoo, Ashley Ziegler, James J. Russo, Wenjia Li, Shenglong Zhang · 发表于：ACS Chemical Biology · 年份：2020 · DOI：10.1021/acschembio.0c00119 · 被引用次数：25 · 研究领域：RNA modifications and cancer、RNA and protein synthesis mechanisms、Cancer-related molecular mechanisms research

Post-transcriptional modifications are intrinsic to RNA structure and function. However, methods to sequence RNA typically require a cDNA intermediate and are either not able to sequence these modifications or are tailored to sequence one specific nucleotide modification only. Interestingly, some of these modifications occur with <100% frequency at their particular sites, and site-specific quantification of their stoichiometries is another challenge. Here, we report a direct method for sequencing tRNA Phe without cDNA by integrating a two-dimensional hydrophobic RNA end-labeling strategy with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNA Phe was sequenced and the identity, location, and stoichiometry of all eleven different RNA modifications was determined, five of which were not 100% modified, including a 2′-O-methylated G (Gm) in the wobble anticodon position as well as an N 2, N 2 -dimethylguanosine (m 2 2 G), a 7-methylguanosine (m 7 G), a 1-methyladenosine (m 1 A), and a wybutosine (Y), suggesting numerous post-transcriptional regulations in tRNA. Two truncated isoforms at the 3′-CCA tail of the tRNA Phe (75 nt with a 3′-CC tail (80% abundance) and 74 nt with a 3′-C tail (3% abundance)) were identified in addition to the full-length 3′-CCA-tailed tRNA Phe (76 nt, 17% abundance). We discovered a new isoform with A–G transitions/editing at the 44 and 45 positions in the tRNA Phe variable loop, and discuss possible mech...