Structural study of human spliceosomal protein SF3b1

• Project/Area Number: 15K06982
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: Musashino University
• Principal Investigator: Kuwasako Kanako 武蔵野大学, 薬学部, 講師 (10568736)
• Co-Investigator(Kenkyū-buntansha): 坂本 泰一 千葉工業大学, 先進工学部, 教授 (40383369) ; 吉村 明 東北医科薬科大学, 薬学部, 講師 (70302164)
• Project Period (FY): 2015-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: スプライシング / がん / pre-mRNAスプライシング / NMR / SELEX / mRNAスプライシング

Outline of Final Research Achievements

Mutations in some specific lysine residues in a spliceosomal protein SF3b1 have often been found in several types of cancer cells. However, it remains unknown not only what part SF3b1 plays in the splicing reaction, but also how the mutations in SF3b affect the process of splicing reaction and contribute to malignant transformation. To elucidate the function of SF3b1 in the process by structural analysis, we attempted to prepare the recombinant SF3b1 protein by overproduction in E. coli, but the yield of the protein was very low. Next, we examined the interactions between spliceosomal proteins by NMR titration experiments, showing that a spliceosomal protein in which some mutations had been reported in cancer cells makes interactions with another spliceosomal protein. Thus, we tried to determine the core structure of the complex composed of the two proteins by NMR. Furthermore, we performed SELEX to elucidate the specificity of the spliceosomal protein that binds to the branch point.

Academic Significance and Societal Importance of the Research Achievements

本実験では，SF3b1と共に機能し，がん細胞での変異が報告されている特定のスプライシング因子が別のスプライシング因子と相互作用することが判明したため，これらの複合体形成部位の立体構造解析を進めている。これらの相互作用メカニズムの解明は，がん化とスプライシング因子の変異との関係を明らかにするのに非常に役立つと考えられる。また，ブランチ部位に結合するスプライシング因子について，RNA配列の特異性の解明を試みており，ヒトにおける保存性の低いブランチ部位配列を認識するメカニズムに関して，新たな知見が得られると考える。

Research Products

• [Journal Article] Solution structure of the first RNA recognition motif domain of human spliceosomal protein SF3b49 and its mode of interaction with a SF3b145 fragment (2017)
  • Author(s): Kuwasako, K.
  • Journal Title: Protein Science Volume: 26 Issue: 2 Pages: 280-290
  • DOI: 10.1002/pro.3080
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Glycolytic flux controls D-serine synthesis through glyceraldehyde-3-phosphate dehydrogenase in astrocytes. (2015)
  • Author(s): Suzuki M, Sasabe J, Miyoshi Y, Kuwasako K, Muto Y, Hamase K, Matsuoka M, Imanishi N, Aiso S.
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 112 Issue: 17
  • DOI: 10.1073/pnas.1416117112
  • Peer Reviewed / Open Access
• [Journal Article] A novel 3' splice site recognition by the two zinc fingers in the U2AF small subunit. (2015)
  • Author(s): Yoshida H, Park S-Y, Oda T, Akiyoshi T, Sato M, Shirouzu M, Tsuda K, Kuwasako K, Unzai S, Muto Y, Urano T, Obayashi E.
  • Journal Title: Genes Dev. Volume: 29 Issue: 15 Pages: 1649-1660
  • DOI: 10.1101/gad.267104.115
  • Peer Reviewed / Open Access
• [Presentation] スプライシング因子の複合体に結合するaptamerの特性解析 (2018)
  • Author(s): 佐藤謙太郎，柳澤拓也，瀧澤将行，天野亮，武藤裕，桑迫香奈子，坂本泰一
  • Organizer: 第41回 日本分子生物学会年会
• [Presentation] スプライシング因子の複合体に結合するaptamerの結合能の解析 (2018)
  • Author(s): 佐藤謙太郎，柳澤拓也，瀧澤将行，天野亮，武藤裕，桑迫香奈子，坂本泰一
  • Organizer: 2018年度 日本生化学会関東支部例会
• [Presentation] Selection of RNA sequences that bind to a complex of splicing factors. (2017)
  • Author(s): 柳澤拓也，桑迫香奈子，天野亮，瀧澤将行，武藤裕，坂本泰一
  • Organizer: 第40回 日本分子生物学会年会
• [Presentation] スプライシング因子の複合体に対するaptamerの取得と解析 (2017)
  • Author(s): 柳澤拓也，桑迫香奈子，徳田正明，天野亮，武藤裕，坂本泰一
  • Organizer: 平成29年度 日本生化学会関東支部例会
• [Presentation] In vitro selection of RNA aptamers to a splicing factor. (2016)
  • Author(s): Takuya Yanagisawa, Kanako Kuwasako, Ryo Amano, Yutaka Muto, Taiichi Sakamoto
  • Organizer: 18th Annual Meeting (21st Annual Meeting of the RNA Society)
  • Place of Presentation: Kyoto International Conference Center
  • Int'l Joint Research
• [Presentation] Solution structures of the two RNA recognition motif (RRM) domains of the human spliceosomal protein SF3b49. (2016)
  • Author(s): Kanako Kuwasako, Kengo Tsuda, Mari Takahashi, Atsuko Sato, Naoya Tochio, Makoto Inoue, Takaho Terada, Takanori Kigawa, Naohiro Kobayashi, Mikako Shirouzu, Takuhiro Ito, Taiichi Sakamoto, Nobukazu Nameki, Kaori Wakamatsu, Peter Guntert, Seizo Takahashi, Shigeyuki Yokoyama, Yutaka Muto
  • Organizer: 18th Annual Meeting (21st Annual Meeting of the RNA Society)
  • Place of Presentation: Kyoto International Conference Center
  • Int'l Joint Research