Identification technique development for post-transcriptional modifications of pseudouridylation in human RNA by mass spectrometry

• Project/Area Number: 18K14253
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 34020:Analytical chemistry-related
• Research Institution: Tokyo Metropolitan University
• Principal Investigator: Yamaki Yuka 首都大学東京, 理学研究科, 客員研究員 (80781525)
• Project Period (FY): 2018-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2019: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2018: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: 質量分析 / RNA / 転写後修飾 / シュードウリジン / LC-MS

Outline of Final Research Achievements

U2 snRNA and U3 snoRNA were purified from RNA biosynthesized with uridine-5,6-d2 in the human TK6 cells deficient in mono phosphate synthase. U2 snRNA and U3 snoRNA were analyzed by LC-MS after digestion with RNase. Spectrum were searched in the database Ariadne, and novel pseudouridylation sites were revealed.

Academic Significance and Societal Importance of the Research Achievements

シュードウリジン化酵素によりウリジンがシュードウリジン化修飾を受けても、化学的性質に変化は見られず質量に差が生じないことから、質量分析計で両者を判別することは困難であった。しかし、ΔUMPS株とラベルウリジンを用いることで、シュードウリジン化修飾により生じる質量差を利用することで、シュードウリジン化サイトを明らかにすることが可能になった。

Research Products

• [Journal Article] Landscape of the complete RNA chemical modifications in the human 80S ribosome. (2018)
  • Author(s): Taoka M, Nobe Y, Yamaki Y, Sato K, Ishikawa H, Izumikawa K, Yamauchi Y, Hirota K, Nakayama H, Takahashi N, Isobe T.
  • Journal Title: Nucleic Acids Res. Volume: 46 Issue: 18 Pages: 9289-9298
  • DOI: 10.1093/nar/gky811
  • Peer Reviewed / Open Access