Identification of the role of transcription termination and polyadenylation in ALS using multiple high-throughput sequencing analysis

2021 Fiscal Year Final Research Report

• Project/Area Number: 18K06058
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 43010:Molecular biology-related
• Research Institution: Nagoya University
• Principal Investigator: Masuda Akio 名古屋大学, 医学系研究科, 准教授 (10343203)
• Project Period (FY): 2018-04-01 – 2022-03-31
• Keywords: RNA結合タンパク / RNA修飾 / RNA代謝 / CLIP / tRIP

Outline of Final Research Achievements

We developed the tRIP method (targeted RNA immunoprecipitation), which enables identification of the protein-RNA interaction sites to a similar level of accuracy as CLIP-seq, but requires only thousands of cells. tRIP requires only 2 days to generate a cDNA library with a single RNA purification step. tRIP adopts exonuclease treatment, and abrogates tedious SDS-PAGE and membrane transfer that are essential in CLIP methodology. High sensitivity of tRIP allows us to perform serial immunoprecipitation to identify the protein-protein-RNA interaction sites starting from a subnuclear fraction.tRIP provides new insights into the regulatory mechanism of co-transcriptional RNA processing by RNA processing factors.

Free Research Field

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Academic Significance and Societal Importance of the Research Achievements

従来困難であった微量検体からのRNA-蛋白結合やRNA修飾部位の解析手法の開発に成功した。従来法を大幅に上回る感度を有しながらも、手技は簡単になっており、今後、RNA-蛋白結合部位解析の標準手法となることが期待される。本手法を利用すれば、従来、少量しか入手できないために解析困難であった、初代継代培養細胞や患者検体内でのRNA代謝様式を解明できることとなり、RNA代謝の生理的意義解明や病態解明の大幅な進歩につながることが期待される。