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

• 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
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2018: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: RNA結合タンパク / RNA修飾 / RNA代謝 / CLIP / tRIP / スプライシング / IDR / SRSF3 / エクソン / 転写制御 / 相分離 / 天然変性領域 / RNA-蛋白相互作用 / FUS / U1 snRNP / RNAP II / MATR3

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.

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。

Research Products

• [Journal Article] Meclozine ameliorates skeletal muscle pathology and increases muscle forces in mdx mice (2022)
  • Author(s): Kawamura Yusuke、Hida Tetsuro、Ohkawara Bisei、Matsushita Masaki、Kobayashi Takeshi、Ishizuka Shinya、Hiraiwa Hideki、Tanaka Satoshi、Tsushima Mikito、Nakashima Hiroaki、Ito Kenyu、Imagama Shiro、Ito Mikako、Masuda Akio、Ishiguro Naoki、Ohno Kinji
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 592 Pages: 87-92
  • DOI: 10.1016/j.bbrc.2022.01.003
  • Peer Reviewed / Open Access
• [Journal Article] Regulated splicing of large exons is linked to phase‐separation of vertebrate transcription factors (2021)
  • Author(s): Kawachi Toshihiko、Masuda Akio、Yamashita Yoshihiro、Takeda Jun‐ichi、Ohkawara Bisei、Ito Mikako、Ohno Kinji
  • Journal Title: The EMBO Journal Volume: 40 Issue: 22
  • DOI: 10.15252/embj.2020107485
  • Peer Reviewed / Open Access
• [Journal Article] Rapidly Growing Protein-Centric Technologies to Extensively Identify Protein?RNA Interactions: Application to the Analysis of Co-Transcriptional RNA Processing (2021)
  • Author(s): Masuda Akio、Kawachi Toshihiko、Ohno Kinji
  • Journal Title: International Journal of Molecular Sciences Volume: 22 Issue: 10 Pages: 5312-5312
  • DOI: 10.3390/ijms22105312
  • Peer Reviewed / Open Access
• [Journal Article] Zonisamide upregulates neuregulin-1 expression and enhances acetylcholine receptor clustering at the in vitro neuromuscular junction (2021)
  • Author(s): Inoue Taro、Ohkawara Bisei、Bushra Samira、Kanbara Shunsuke、Nakashima Hiroaki、Koshimizu Hiroyuki、Tomita Hiroyuki、Ito Mikako、Masuda Akio、Ishiguro Naoki、Imagama Shiro、Ohno Kinji
  • Journal Title: Neuropharmacology Volume: 195 Pages: 108637-108637
  • DOI: 10.1016/j.neuropharm.2021.108637
  • Peer Reviewed / Open Access
• [Journal Article] tRIP-seq法を用いた微量検体からのRNA修飾部位検出 (2021)
  • Author(s): 増田章男、河地利彦、大野欽司
  • Journal Title: 細胞 Volume: 53 Pages: 50-52
  • NAID: 40022799109
• [Journal Article] tRIP-seq Reveals Repression of Premature Polyadenylation by Co-Transcriptional FUS-U1 snRNP Assembly (2020)
  • Author(s): Akio Masuda, Toshihiko Kawachi, Jun-Ichi Takeda, Bisei Ohkawara, Mikako Ito, Kinji Ohno
  • Journal Title: EMBO Rep. Volume: - Issue: 5
  • DOI: 10.15252/embr.201949890
  • NAID: 120006884959
  • Peer Reviewed / Open Access
• [Journal Article] Gene Expression Profile at the Motor Endplate of the Neuromuscular Junction of Fast-Twitch Muscle (2020)
  • Author(s): Huang Kun、Li Jin、Ito Mikako、Takeda Jun-Ichi、Ohkawara Bisei、Ogi Tomoo、Masuda Akio、Ohno Kinji
  • Journal Title: Frontiers in Molecular Neuroscience Volume: 13 Pages: 154-154
  • DOI: 10.3389/fnmol.2020.00154
  • Peer Reviewed / Open Access
• [Journal Article] CTGF/CCN2 facilitates LRP4‐mediated formation of the embryonic neuromuscular junction (2020)
  • Author(s): Ohkawara Bisei、Kobayakawa Akinori、Kanbara Shunsuke、Hattori Takako、Kubota Satoshi、Ito Mikako、Masuda Akio、Takigawa Masaharu、Lyons Karen M、Ishiguro Naoki、Ohno Kinji
  • Journal Title: EMBO reports Volume: 21 Issue: 8
  • DOI: 10.15252/embr.201948462
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Ubiquitination of DNA Damage-Stalled RNAPII Promotes Transcription-Coupled Repair (2020)
  • Author(s): Nakazawa Y, Hara Y, Oka Y, Komine O, van den Heuvel D, Guo C, Daigaku Y, Isono M, He Y, Shimada M, Kato K, Jia N, Hashimoto S, Kotani Y, Miyoshi Y, Tanaka M, Sobue A, Mitsutake N, Suganami T, Masuda A, Ohno K, Nakada S, Mashimo T, Yamanaka K, Luijsterburg MS, Ogi T
  • Journal Title: Cell Volume: 19 Issue: 6 Pages: 1228-1244
  • DOI: 10.1016/j.cell.2020.02.010
  • NAID: 120006871293
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Inhibition of cyclooxygenase-1 by nonsteroidal anti-inflammatory drugs demethylates MeR2 enhancer and promotes Mbnl1 transcription in myogenic cells. (2020)
  • Author(s): Huang K, Masuda A, Chen G, Bushra S, Kamon M, Araki T, Kinoshita M, Ohkawara B, Ito M, Ohno K
  • Journal Title: Sci Rep Volume: 10 Issue: 1 Pages: 2558-2558
  • DOI: 10.1038/s41598-020-59517-y
  • Peer Reviewed / Open Access
• [Journal Article] Mianserin suppresses R-spondin 2-induced activation of Wnt/β-catenin signaling in chondrocytes and prevents cartilage degradation in a rat model of osteoarthritis (2019)
  • Author(s): Okura Toshiaki、Ohkawara Bisei、Takegami Yasuhiko、Ito Mikako、Masuda Akio、Seki Taisuke、Ishiguro Naoki、Ohno Kinji
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 2808-2808
  • DOI: 10.1038/s41598-019-39393-x
  • Peer Reviewed / Open Access
• [Journal Article] Differential effects of spinal motor neuron-derived and skeletal muscle-derived Rspo2 on acetylcholine receptor clustering at the neuromuscular junction (2018)
  • Author(s): Li Jin、Ito Mikako、Ohkawara Bisei、Masuda Akio、Ohno Kinji
  • Journal Title: Scientific Reports Volume: 8 Issue: 1 Pages: 13577-13577
  • DOI: 10.1038/s41598-018-31949-7
  • Peer Reviewed / Open Access
• [Journal Article] Lack of Fgf18 causes abnormal clustering of motor nerve terminals at the neuromuscular junction with reduced acetylcholine receptor clusters (2018)
  • Author(s): Ito Kenyu、Ohkawara Bisei、Yagi Hideki、Nakashima Hiroaki、Tsushima Mikito、Ota Kyotaro、Konishi Hiroyuki、Masuda Akio、Imagama Shiro、Kiyama Hiroshi、Ishiguro Naoki、Ohno Kinji
  • Journal Title: Sceintific Reports Volume: 8 Issue: 1 Pages: 434-434
  • DOI: 10.1038/s41598-017-18753-5
  • Peer Reviewed / Open Access
• [Journal Article] MYRF is associated with encephalopathy with reversible myelin vacuolization (2018)
  • Author(s): Kurahashi Hirokazu、Azuma Yoshiteru、Masuda Akio、Okuno Tatsuya、Nakahara Eri、Imamura Takuji、Saitoh Makiko、Mizuguchi Masashi、Shimizu Toshiaki、Ohno Kinji、Okumura Akihisa
  • Journal Title: Annals of Neurology Volume: 83 Issue: 1 Pages: 98-106
  • DOI: 10.1002/ana.25125
  • Peer Reviewed / Open Access
• [Presentation] Identification of the nuclear speckles enriched with MATR3, which functionally regulate alternative splicing through spatial compartmentalization by LLPS (2021)
  • Author(s): Takaaki Okamoto, Akio Masuda, Kinji Ohno
  • Organizer: 第44回日本分子生物学会年会
• [Presentation] 増田章男、岡本喬明、大野欽司 (2021)
  • Author(s): RNA結合タンパクMatrin3が形成する核内顆粒による遺伝子発現制御
  • Organizer: 第7回日本筋学会学術集会
  • Invited
• [Presentation] 長いエクソンは天然変性領域をコードしており、SRSF3により安定的にスプライスされる (2020)
  • Author(s): 増田章男 河地利彦 大野欽司
  • Organizer: 第43回日本分子生物学会年会
• [Presentation] The splicing mechanism that secures large exons encoding intrinsic disordered regions of transcription factors (2020)
  • Author(s): Toshihiko Kawachi Akio Masuda, Jun-ichi Takeda, Mikako Ito, Tomonari Hamaguchi, Kinji Ohno
  • Organizer: Experimental Biology 2020 Meeting
• [Presentation] tRIP法による、RNA polymerase IIによる転写中pre-mRNA上での、RNA-protein相互作用部位の同定 (2019)
  • Author(s): Akio Masuda, Toshihiko Kawachi, Junichi Takeda, Kinji Ohno
  • Organizer: 第42回日本分子生物学会年会
• [Presentation] 新規RNA免疫沈降法(tRIP法）による、FUS－U1 snRNP依存性 転写終結制御の解明 (2018)
  • Author(s): Akio Masuda, Toshihiko Kawachi, Junichi Takeda, Kinji Ohno
  • Organizer: 第41回 日本分子生物学会年会
• [Remarks] Tracing mechanisms of large exon splicing
  • URL: https://www.eurekalert.org/news-releases/935542
• [Remarks] the regulation of large exon splicing is critical
  • URL: https://en.nagoya-u.ac.jp/research/activities/news/2021/11/20211118-01.html
• [Remarks] 転写ネットワーク発展の礎となったゲノム進化を同定
  • URL: https://www.med.nagoya-u.ac.jp/medical_J/research/pdf/EMBO_211004.pdf
• [Remarks] 名古屋大学大学院医学系研究科神経遺伝情報公式HP
  • URL: https://www.med.nagoya-u.ac.jp/neurogenetics/
• [Remarks] 名古屋大学大学院医学系研究科神経遺伝情報公式HP
  • URL: https://www.med.nagoya-u.ac.jp/medical_J/laboratory/neurological-diseases-cancer/dept-neuroscience/neurogenetics/