Mechanism of the ribonucleoprotein complex formation of SARS-CoV2 N protein

• Project/Area Number: 21H02438
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 43040:Biophysics-related
• Research Institution: Tohoku University
• Principal Investigator: Takahashi Satoshi 東北大学, 多元物質科学研究所, 教授 (30283641)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥17,550,000 (Direct Cost: ¥13,500,000、Indirect Cost: ¥4,050,000); Fiscal Year 2023: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2022: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2021: ¥7,410,000 (Direct Cost: ¥5,700,000、Indirect Cost: ¥1,710,000)
• Keywords: SARS-Cov2 / Nタンパク質 / gRNA / 一分子蛍光分光法 / 蛍光相関分光法 / SARS-CoV2 / 新型コロナウイルス / ヌクレオカプシドタンパク質 / ヌクレオカプシッドタンパク質N / 蛍光顕微鏡観察 / ヌクレオカプシッドタンパク質

Outline of Research at the Start

本計画では、SARS-Cov2のヌクレオカプシドタンパク質（N）が、ウイルスのゲノムRNAと液滴や複合体を作る分子機構の解明を目指す。Nは宿主内でゲノムRNAと集合して液滴を形成した後に、gRNAが一個ずつ選別されRNPが形成されるというウイルス複製機構が提案されている。本研究では、一分子蛍光分光法と一分子蛍光顕微鏡観察を駆使し、i）NとgRNAによる液液相分離機構の解明、ii）NとgRNAによるRNPのin vitro構成法の確立、iii）NとgRNAによるRNP形成過程の観察、などを実施する。さらに、iv）Nの小分子阻害剤がこれらの過程に与える影響を調べる。

Outline of Final Research Achievements

Interaction between N and RNA fragments with stem-loop and single-stranded structures was investigated. A method for the purification of the N protein was established. The single-molecule FRET spectroscopy and fluorescence correlation spectroscopy showed that N bind with RNA samples strongly irrespective of their secondary structures, and that N attracts two RNA fragments. The results suggest that the function of N is to bind and to attract two parts of the long gRNA and help its compaction. Experimental methods such as fluorescence correlation spectroscopy and single molecule fluorescence spectroscopy have been used to investigate the structure of long RNAs.

Academic Significance and Societal Importance of the Research Achievements

本研究を実施することで、コロナウイルスの複製に必須の働きをもつNタンパク質の機能をより正確に理解することが可能となった。さらには、gRNAとNタンパク質が結合することで、gRNAがフォールディングを起こしてRNPを形成するgRNAのフォールディング仮説を提案した。この仮説を今後証明できれば、ウイルスの成熟過程を阻害するための効果的な方法を提案できる可能性がある。

Research Products

• [Journal Article] Simple and Efficient Detection Scheme of Two-Color Fluorescence Correlation Spectroscopy for Protein Dynamics Investigation from Nanoseconds to Milliseconds (2023)
  • Author(s): Sano Yutaka、Itoh Yuji、Kamonprasertsuk Supawich、Suzuki Leo、Fukasawa Atsuhito、Oikawa Hiroyuki、Takahashi Satoshi
  • Journal Title: ACS Physical Chemistry Au Volume: 4 Issue: 1 Pages: 85-93
  • DOI: 10.1021/acsphyschemau.3c00040
  • Peer Reviewed / Open Access
• [Journal Article] Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis (2023)
  • Author(s): Han Yibing、Tomita Takeshi、Kato Masayoshi、Ashihara Norihiro、Higuchi Yumiko、Matoba Hisanori、Wang Weiyi、Hayashi Hikaru、Itoh Yuji、Takahashi Satoshi、Kurita Hiroshi、Nakayama Jun、Okumura Nobuo、Hiratsuka Sachie
  • Journal Title: Nature Communications Volume: 14 Issue: 1 Pages: 4960-4960
  • DOI: 10.1038/s41467-023-40371-1
  • Peer Reviewed
• [Journal Article] Flexible Target Recognition of the Intrinsically Disordered DNA-Binding Domain of CytR Monitored by Single-Molecule Fluorescence Spectroscopy (2022)
  • Author(s): Mitra Shrutarshi、Oikawa Hiroyuki、Rajendran Divya、Kowada Toshiyuki、Mizukami Shin、Naganathan Athi N.、Takahashi Satoshi
  • Journal Title: The Journal of Physical Chemistry B Volume: 126 Issue: 33 Pages: 6136-6147
  • DOI: 10.1021/acs.jpcb.2c02791
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Hyper-mobile Water and Raman 2900 cm^?1 Peak Band of Water Observed around Backbone Phosphates of Double Stranded DNA by High-Resolution Spectroscopies and MD Structural Feature Analysis of Water (2022)
  • Author(s): Suzuki Makoto、Tsuchiko Akira、Tanaka Yoshiyuki、Matubayasi Nobuyuki、Mogami George、Uozumi Nobuyuki、Takahashi Satoshi
  • Journal Title: The Journal of Physical Chemistry B Volume: 127 Issue: 1 Pages: 285-299
  • DOI: 10.1021/acs.jpcb.2c06952
  • Peer Reviewed
• [Journal Article] Quantification of Entropic Excluded Volume Effects Driving Crowding-Induced Collapse and Folding of a Disordered Protein (2022)
  • Author(s): Rajendran Divya、Mitra Shrutarshi、Oikawa Hiroyuki、Madhurima Kulkarni、Sekhar Ashok、Takahashi Satoshi、Naganathan Athi N.
  • Journal Title: The Journal of Physical Chemistry Letters Volume: 13 Issue: 13 Pages: 3112-3120
  • DOI: 10.1021/acs.jpclett.2c00316
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Molecular principles of recruitment and dynamics of guest proteins in liquid droplets (2021)
  • Author(s): Kamagata Kiyoto、Iwaki Nanako、Hazra Milan Kumar、Kanbayashi Saori、Banerjee Trishit、Chiba Rika、Sakomoto Seiji、Gaudon Virginie、Castaing Bertrand、Takahashi Hiroto、Kimura Michiko、Oikawa Hiroyuki、Takahashi Satoshi、Levy Yaakov
  • Journal Title: Scientific Reports Volume: 11 Issue: 1 Pages: 19323-19323
  • DOI: 10.1038/s41598-021-98955-0
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Extracellular mRNA transported to the nucleus exerts translation-independent function (2021)
  • Author(s): Tomita Takeshi、Kato Masayoshi、Mishima Taishi、Matsunaga Yuta、Sanjo Hideki、Ito Ken-ichi、Minagawa Kentaro、Matsui Toshimitsu、Oikawa Hiroyuki、Takahashi Satoshi、Takao Toshifumi、Iwai Noriki、Mino Takashi、Takeuchi Osamu、Maru Yoshiro、Hiratsuka Sachie
  • Journal Title: Nature Communications Volume: 12 Issue: 1 Pages: 3655-3655
  • DOI: 10.1038/s41467-021-23969-1
  • Peer Reviewed / Open Access
• [Presentation] Conformational elucidation of SARS-CoV2 genomic RNA elements by single-molecule FRET measurements (2023)
  • Author(s): Shrutarshi Mitra, Yuji Itoh, Takuya Katayama, Satoshi Takahashi
  • Organizer: 第61回日本生物物理学会年会
• [Presentation] ウイルスRNA構造の理解のためのRNA構造の測定と推定 (2023)
  • Author(s): 伊藤 優志 , 片山 拓也 , 高橋 聡
  • Organizer: 第61回日本生物物理学会年会
• [Presentation] 全光子記録方式による二色蛍光相関分光法：ナノ秒からミリ秒領域におけるタンパク質ダイナミクスの観測 (2023)
  • Author(s): 佐野豊, 伊藤優志, 小井川浩之, 高橋聡
  • Organizer: 第61回日本生物物理学会年会
• [Presentation] SARS-CoV-2スパイクタンパク質の二重蛍光ラベル化とラベル化試料の一分子蛍光分光法による構造ダイナミクスの解明 (2023)
  • Author(s): 森 大晟, 伊藤 優志, 鈴木 干城, 橋口 隆生, 高橋 聡
  • Organizer: 第61回日本生物物理学会年会
• [Presentation] 一分子蛍光測定を用いたSARS-CoV-2 Nタンパク質とRNAの結合様式の解明 (2023)
  • Author(s): 金田 直也, 遠藤 隼, 鈴木 怜和, 伊藤 優志, 小井川 浩之, 高橋 聡
  • Organizer: 第61回日本生物物理学会年会
• [Presentation] Single-molecule FRET and FCS investigation of the interaction between SARS-CoV2 N protein and RNA: Does the formation of ribonucleoprotein granule resemble protein folding? (2023)
  • Author(s): Satoshi Takahashi
  • Organizer: 8th Asian Spectroscopy Conference 2023
  • Int'l Joint Research / Invited
• [Presentation] ポストAlphaFold時代のフォールディング研究 (2023)
  • Author(s): 髙橋 聡
  • Organizer: 日本生物物理学会東北支部会
  • Invited
• [Presentation] タイムタグ光子測定方式によるナノ秒蛍光相関分光測定システムの開発 (2023)
  • Author(s): 佐野豊, 伊藤優志, 小井川浩之, 高橋聡
  • Organizer: 日本生物物理学会東北支部会
• [Presentation] AlphaFold時代にこそ重要性を増すアンサンブルおよび一分子蛍光分光法の基礎 (2022)
  • Author(s): 髙橋 聡
  • Organizer: 第22回日本蛋白質科学会年会
  • Invited
• [Presentation] タイムタグ光子測定方式によるナノ秒蛍光相関分光測定システムの開発 (2022)
  • Author(s): 佐野豊, 伊藤優志, 小井川浩之, 高橋聡
  • Organizer: 第60回日本生物物理学会年会
• [Presentation] 一分子蛍光測定を目指した SARS-CoV2 の N 蛋白質の精製及びラベル化 (2022)
  • Author(s): 遠藤隼, 伊藤優志, 小井川浩之, 高橋聡
  • Organizer: 第60回日本生物物理学会年会
• [Presentation] Single molecule fluorescence investigations on the structure transitions of LAF1- RGG upon the RNA binding and the droplet formation (2021)
  • Author(s): Kanna Fujita, Michiko Kimura, Hiroto Takahashi, Satoshi Takahashi, Hiroyuki Oikawa
  • Organizer: The 59th Annual Meeting of the Biophysical Society
• [Presentation] Structural Characterization of RNA upon the binding with SARS-CoV-2 N Protein by Single Molecule Fluorescence Measurements (2021)
  • Author(s): Leo Suzuki, Hiroyuki Oikawa, Satoshi Takahashi
  • Organizer: The 59th Annual Meeting of the Biophysical Society
• [Presentation] Conformational dynamics of E. coil Cytidine Repressor DNA Binding domain studied by Single-molecule Fluorescence Spectroscopy (2021)
  • Author(s): Shrutarshi Mitra, Hiroyuki Oikawa, Divya Rajendran, Athi N.Naganathan, Satoshi Takahashi
  • Organizer: The 59th Annual Meeting of the Biophysical Society