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Molecular biological studies on temporal and spatial regulation of translation during oocyte maturation in mouse

武井, 夏海 北海道大学

2021.03.25

概要

卵巣において卵母細胞は減数分裂を第一分裂前期で停止した未成熟卵として存在し、その後の減数分裂や発生に必要なタンパク質のmRNAを、翻訳を抑制した状態で卵細胞質に蓄積する。ホルモン等の刺激を受けると未成熟卵は減数分裂を再開し、第二分裂中期で再び停止する。この過程は卵成熟と呼ばれ、卵母細胞は受精能を獲得した成熟卵となる。卵成熟は卵細胞質に蓄積したmRNAが時期特異的に翻訳されることで進行する。減数分裂第一分裂では、数百種類のmRNAが翻訳を制御されることがマウスやゼブラフィッシュ、カエルの卵を使った研究で知られる。一方第二分裂では、千種類以上のmRNAが翻訳を制御される。卵成熟が正しく進行するためには、これらのmRNAの翻訳時期が厳密に制御されることが極めて重要である。現在までに、卵成熟における翻訳制御メカニズムとしてcytoplasmic polyadenylation element-binding protein(CPEB)を介したポリA鎖の伸長がmRNAの翻訳を活性化することが明らかとなっている。しかし千以上のmRNAがCPEBの結合配列をもち、それぞれが卵成熟の異なる時期に翻訳されることから、個々のmRNAの翻訳時期を細かく制御するメカニズムが別に存在すると考えられる。しかし数多くの母性mRNAの翻訳時期を制御するメカニズムは不明な点が多いままである。

 先行研究において、第一分裂で翻訳され、卵成熟の進行に重要な因子であるCyclin B1 mRNAがゼブラフィッシュとマウスの未成熟卵でRNA顆粒を形成すること、この顆粒構造は翻訳が活性化する時期に消失することが明らかとなった。さらに、Cyclin B1 RNA顆粒の形成にはPumilio1(Pum1)というRNA結合タンパク質が必要であることが示された。このことから、Pum1は顆粒の形成と消失を介してターゲットmRNAの翻訳時期を制御することが考えられた。

 本研究では、卵成熟における時期特異的な翻訳制御メカニズムを解明することを目指した。まずChapter Iにおいて、チラミドシグナル増幅システムを用いてRNAを高感度かつ高解像度で検出する手法を確立した。Chapter IIでは、減数分裂第一分裂における翻訳制御メカニズムの解明を目指した。In situ hybridization法によって、Cyclin B1 mRNAと同じく第一分裂で翻訳されるMad2 mRNAがマウスの卵細胞質で顆粒構造を形成し、顆粒の形成と消失によって翻訳が制御されることが示唆された。さらに免疫沈降法とRT-PCRによって、Mad2 mRNAはCyclin B1 mRNAと同じくPum1の標的mRNAであることが明らかとなった。次に、蛍光免疫染色法とGFPを結合させたPum1のコンストラクトによって、Pum1が卵細胞質において凝集体を形成することが示された。この凝集体は卵母細胞においてMad2とCyclin B1 RNA顆粒を囲うように分布した。また、Pum1は卵成熟に伴い凝集体の構造が崩壊し、固相様の性質から液相様の性質に変化することが明らかとなった。Pum1凝集体の構造が維持されるとターゲットmRNAの翻訳並びに卵成熟が抑制されたことから、Pum1の性質の]変化がターゲットmRNAの翻訳を制御し、卵成熟を進行させることが示された。これらのことから、減数分裂第一分裂において、Pum1凝集体の形成と崩壊がターゲットmRNAの翻訳を時空間的に制御することが考えられた。

 Chapter IIIでは、減数分裂第二分裂における翻訳制御メカニズムの解明を目指した。第二分裂で翻訳が開始するEmi2 mRNAは、異なる時期に翻訳されると減数分裂の停止や単為発生が引き起こされることから、翻訳時期が厳密に制御されることは卵成熟が正しく進行するために極めて重要である。In situ hybridization法により、Emi2 mRNAは卵細胞質でCyclin B1 RNA顆粒とは異なる分布を示す顆粒構造をとることが明らかとなった。さらにEmi2 mRNAの翻訳が活性化する時期に顆粒構造が消失することから、顆粒を介した翻訳制御を受けること示唆された。顆粒構造を形成することから、Cyclin B1 mRNAと同じくEmi2 mRNAはPum1のターゲットmRNAであると考えられたが、免疫沈降法とRT-PCRの結果、Emi2 mRNAにはPum1が結合しないことが明らかとなった。一方、ストレス顆粒や神経顆粒といった他のRNA顆粒の構成タンパク質として知られるHuRとHuBはEmi2とCyclin B1 mRNAに共通して結合した。次にEmi2 mRNAに特異的に結合するタンパク質が存在すると考え、Emi2 mRNAに結合するタンパク質を網羅的に解析した。その結果、Emi2 mRNAに特異的に結合し、mRNAの翻訳制御や安定化に関わるタンパク質が10種類検出された。このうち、Tudor domain-containing protein 3(Tdrd3)はストレス顆粒の構成タンパク質として知られ、ウェスタンブロッティングとRT-PCRによってマウスの卵巣と卵母細胞においてmRNAとタンパク質が検出された。UVクロスリンクと超解像顕微鏡を用いたタンパク質とmRNAの同時検出により、Tdrd3はCyclin B1 mRNAには結合せず、Emi2 mRNAに特異的に結合することが明らかとなった。さらに、α-Tdrd3抗体を卵母細胞に微量注入してから減数分裂を再開させると、第二分裂においてEmi2 mRNAの翻訳開始が遅れたことから、Tdrd3はEmi2 mRNAの翻訳時期の制御に関わることが示唆された。

 本研究により、卵成熟におけるmRNAの翻訳は、これまで示されていたCPEBを介したポリA鎖の伸長による制御に加え、RNA顆粒を介して翻訳時期が制御されることが考えられた。このRNA顆粒は、各mRNAに共通して結合するタンパク質が構造の基盤となることが考えられる。さらに、mRNAによって顆粒を構成するタンパク質の種類が異なることが明らかとなった。第一分裂で翻訳されるCyclin B1とMad2 mRNAはPum1が結合し、Pum1凝集体の性質の変化がRNA顆粒を介した翻訳制御に重要であった。第二分裂では、Emi2 mRNAに特異的に結合し、翻訳時期の制御に関わるタンパク質としてTdrd3が新たに示された。このようにmRNAに結合するタンパク質の種類の違いとRNA顆粒によって卵成熟における翻訳が時間的、空間的に制御されると考えられた。

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