351. Direct Sequencing of tRNA by 2D-HELS-AA MS Seq Reveals Its Different Isoforms and Dynamic Base Modifications
- Author
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Barney Yoo, Wenhao Ni, Xuanting Wang, Wenjia Li, Jiachen Duan, Shundi Shi, Ning Zhang, Ashley Ziegler, James J. Russo, Tony Z. Jia, Shenglong Zhang, and Xiaohong Yuan
- Subjects
0301 basic medicine ,Gene isoform ,Computational biology ,01 natural sciences ,Biochemistry ,Article ,Mass Spectrometry ,Base (group theory) ,03 medical and health sciences ,Isomerism ,RNA, Transfer ,Complementary DNA ,Nucleic acid structure ,RNA Processing, Post-Transcriptional ,Base Pairing ,Sequence (medicine) ,010405 organic chemistry ,Chemistry ,Sequence Analysis, RNA ,RNA ,General Medicine ,0104 chemical sciences ,030104 developmental biology ,Transfer RNA ,Molecular Medicine ,Hydrophobic and Hydrophilic Interactions ,Function (biology) ,Algorithms - Abstract
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 aretailored to sequence one specific nucleotide modification only. Interestingly, some of these modifications occur with Phe without cDNA by integrating atwo-dimensional hydrophobic RNA end-labeling strategy with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNAPhe 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 N2, N2-dimethylguanosine (m22G), a7-methylguanosine (m7G), a1-methyladenosine (m1A), and awybutosine (Y), suggesting numerous post-transcriptional regulations in tRNA. Two truncated isoforms at the 3'-CCA tail of the tRNAPhe (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 tRNAPhe (76 nt, 17% abundance). We discovered a new isoform with A-G transitions/editing at the 44 and 45 positions in the tRNAPhe variable loop, and discuss possible mechanisms related to the emergence and functions of the isoforms with these base transitions or editing. Our method revealed new isoforms, base modifications, and RNA editing as well as their stoichiometries in the tRNA that cannot be determined by current cDNA-based methods, opening new opportunities in the field of epitranscriptomics.
- Published
- 2020