Regulation of CCR4-NOT complex deadenylase activity and cellular responses by MK2-dependent phosphorylation of CNOT2

CCR4-NOT complex-mediated mRNA deadenylation serves critical functions in multiple biological processes, yet how this activity is regulated is not fully understood. Here, we show that osmotic stress induces MAPKAPK-2 (MK2)-mediated phosphorylation of CNOT2. Programmed cell death is greatly enhanced by osmotic stress in CNOT2-depleted cells, indicating that CNOT2 is responsible for stress resistance of cells. Although wild-type (WT) and non-phosphorylatable CNOT2 mutants reverse this sensitivity, a phosphomimetic form of CNOT2, in which serine at the phosphorylation site is replaced with glutamate, does not have this function. We also show that mRNAs have elongated poly(A) tails in CNOT2-depleted cells and that introduction of CNOT2 WT or a non-phosphorylatable mutant, but not phosphomimetic CNOT2, renders their poly(A) tail lengths comparable to those in control HeLa cells. Consistent with this, the CCR4-NOT complex containing phosphomimetic CNOT2 exhibits less deadenylase activity than that containing CNOT2 WT. These data suggest that CCR4-NOT complex deadenylase activity is regulated by post-translational modification, yielding dynamic control of mRNA deadenylation

ARE-binding protein ZFP36L1 interacts with CNOT1 to directly repress translation via a deadenylation-independent mechanism

Eukaryotic gene expression can be spatiotemporally tuned at the post-transcriptional level by cis-regulatory elements in mRNA sequences. An important example is the AU-rich element (ARE), which induces mRNA destabilization in a variety of biological contexts in mammals and can also mediate translational control. Regulation is mediated by trans-acting factors that recognize the ARE, such as Tristetraprolin (TTP) and BRF1/ZFP36L1. Although both proteins can destabilize their target mRNAs through the recruitment of the CCR4-NOT deadenylation complex, TTP also directly regulates translation. Whether ZFP36L1 can directly repress translation remains unknown. Here, we used an in vitro translation system derived from mammalian cell lines to address this key mechanistic issue in ARE regulation by ZFP36L1. Functional assays with mutant proteins reveal that ZFP36L1 can repress translation via AU-Rich elements independent of deadenylation. ZFP36L1-mediated translation repression requires interaction between ZFP36L1 and CNOT1, suggesting that it might use a repression mechanism similar to either TPP or miRISC. However, several lines of evidence suggest that the similarity ends there. Unlike, TTP, it does not efficiently interact with either 4E-HP or GIGYF2, suggesting it does not repress translation by recruiting these proteins to the mRNA cap. Moreover, ZFP36L1 could not repress ECMV-IRES driven translation and was resistant to pharmacological eIF4A inhibitor silvestrol, suggesting fundamental differences with miRISC repression via eIF4A. Collectively, our results reveal that ZFP36L1 represses translation directly and suggest that it does so via a novel mechanism distinct from other translational regulators that interact with the CCR4-NOT deadenylase complex

The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function

Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis

Ownership structure, investment behaviour and firm performance in Japanese manufacturing industries

Abstract Using data spanning the 1996-98 fiscal years of 247 of Japan's largest manufacturers, we empirically evaluate the extent to which a firm's investment behaviour and financial performance are influenced by its ownership structure. To do so, we examine six distinct categories of Japanese shareholders: foreign investors, investment funds, pension funds, banks and insurance companies, affiliated companies and insiders. Our findings strongly indicate that the relationship between the equity stakes of a particular category of investor and a firm's financial performance and investment behaviour is considerably more complex than is depicted in simple principal-agent representations. Such a result emphasizes the importance of making finely grained and contextually relevant distinctions when modelling and evaluating corporate governance relations

食品製造における微生物管理に関する研究

第1章 序論 / p1 第2章 蒲鉾のPseudomonas sp.汚染について / p7 　第1節 蛍光性軟化変敗原因菌の分離と同定 / p7 　　1.変敗菌の分離と同定 / p7 　　2.分離株の世代交代時間と増殖温度 / p19 　第2節 分離株の蒲鉾軟化再現実験 / p21 　第3節 市販蒲鉾の微生物汚染状況 / p25 　　1.市販蒲鉾の保存試験と変敗菌の分離同定 / p25 　　2.分離菌による蒲鉾軟化再現実験 / p29 　第4節 蛍光性Pseudomonas sp.簡易試験用培地の検討 / p30 　　1.蛍光性Pseudomonas sp.簡易試験用培地の処方決定 / p30 　　2.蛍光性Pseudomonas sp.簡易試験用培地の精度検定 / p40 　　3.考察 / p43 　第5節 蒲鉾製造工程の微生物汚染状況 / p44 　　1.蒲鉾製造工程の拭き取り試験と検出菌の同定 / p44 　　2.検出菌による蒲鉾軟化の再現実験 / p47 　第6節 包装蒲鉾の汚染メカニズム / p47 　第7節 蒲鉾および製造行程より分離されたPseudomonas sp.のプロテアーゼ産生能 / p50 第3章 蒲鉾表面でのPseudomonas sp.の増殖について / p56 　第1節 低分子N源が増殖に及ぼす影響 / p56 　第2節 プロテアーゼ処理が増殖に及ぼす影響 / p58 　第3節 各種粉末たん白が増殖に及ぼす影響 / p60 　　1.粉末たん白ゲル上での増殖 / p60 　　2.蒲鉾上での増殖 / p62 　　3.ポークゲル上での増殖 / p63 　　4.ロースハム上での増殖 / p64 　　5.牛血漿たん白がp.fluorescensの増殖に及ぼす影響 / p67 　6.考察 / p67 　第4節 K-1株が産生する菌体外プロテアーゼの性質 / p69 　　1.各種化合物が活性に及ぼす影響 / p69 　　2.食品添加物が活性に及ぼす影響 / p73 　　3.トリポリリン酸ナトリウムがK-1株の増殖に及ぼす影響 / p73 　　4.考察 / p77 第4章 ホップの抗菌効果について / p78 　第1節 ホップエキスの抗菌カ / p78 　第2節 ホップエキスと制菌素材の併用による大腸菌への抗菌効果 / p81 　第3節 ホップエキスとメタリン酸ナトリウムの併用による大腸菌への抗菌効果 / p83 　　1.大腸菌への抗菌効果 / p83 　　2.併用系での各試料濃度の影響 / p87 　　3.併用系での試料の添加時期の影響 / p88 　　4.併用系での試料の添加順序と濃度の影響 / p94 　　5.試料接触処理菌の増殖能 / p98 　　6.試料接触処理による菌体成分の漏洩 / p101 　　7.マッシュポテトでの併用効果 / p101 　　8.考察 / p104 　第4節 ホップエキスと制菌素材の併用によるグラム陰性菌への抗菌効果 / p106 　　1.ホップエキスとメタリン酸ナトリウムの併用効果 / p106 　　2.ホップエキスと酢酸ナトリウムの併用効果 / p109 　　3.ホップエキスとグリシンの併用効果 / p110 　　4.考察 / p112 　第5節 微生物の耐熱性に及ぼすホップエキスの影響 / p114 　　1.セレウス菌芽胞の耐熱性に及ぼす影響 / p114 　　2.大腸菌の耐熱性に及ぼす影響 / p114 　　3.考察 / p116 　第6節 大腸菌の凍結・解凍耐性に及ぼすホップエキスの影響 / p119 　　1.インビトロ系での凍結耐性に及ぼす影響 / p119 　　2.豚ミンチ肉系での凍結耐性に及ぼす影響 / p125 　　3.蒲鉾での凍結耐性に及ぼす影響 / p127 　　4.考察 / p130 　第7節 ホップエキスの熱安定性 / p131 　　1.ホップエキスの抗菌力に及ぼす加熱温度の影響 / p131 　　2.オートクレーブ処理がホップエキスの抗菌力に及ぼす影響 / p133 　第8節 ホップエキスの抗菌力に及ぼす各種成分の影響 / p135 　　1.代表的な食品成分の影響 / p135 　　2.金属イオンの影響 / p138 　　3.考察 / p140 　第9節 水素添加ホップエキスの抗菌効果 / p140 　　1.水素添加ホップエキスの抗菌スペクトル / p140 　　2.水素添加ホップエキスと制菌素材との併用によるグラム陰性菌への抗菌効果 / p148 　　3.考察 / p152 　第10節 ホップエキスのアルコール製剤および洗浄除菌剤への配合 / p153 　　1.殺菌力への影響 / p153 　　2.ホップエキス配合アルコール製剤の食品への利用 / p156 　　3.考察 / p164 　第11節 食品での保存性向上効果 / p167 　　1.ホップエキスの粉末化 / p167 　　2.ホップエキス粉末の抗菌力 / p171 　　3.食品ホモジネイト系での保存性向上効果 / p172 　　4.食品での保存性向上効果 / p179 　　5.考察 / p186 第5章 総合考察 / p189 謝辞 / p200 引用文献 / p201広島大学(Hiroshima University)博士(農学)Agriculturedoctora