翻译起始的分析在应力条件由多聚核糖体剖析
Summary
在这里，我们描述一个变化来分析真核细胞中的mRNA的翻译的响应于胁迫条件的启动方法。此方法是基于换算从非翻译的核糖体的核糖体的蔗糖梯度的速度分离。
Abstract
mRNA翻译的精确控制是基本的真核细胞内环境稳定，特别是在应对生理和病理的压力。这个方案的改变可导致受损细胞的生长，癌症发展的一个标志，或过早的细胞死亡如见于神经变性疾病。多的关于对翻译控制的分子基础所谓已经从多核糖体分析使用密度梯度分级分离系统获得的。这种技术依赖于细胞质提取物对超速的线性蔗糖梯度。一旦旋转完成时，该系统允许分馏对应于不同翻译离心分离区与定量核糖体种群，从而导致多核糖体模式。变化多核糖体轮廓是指示发生在响应各类应力的翻译起始的变化或缺陷。此技术还允许以评估日特定蛋白对翻译起始é角色，并衡量特定mRNA的翻译活动。这里我们描述的协议，以便评估在正常或胁迫生长条件下的真核细胞和组织的翻译起始执行多核糖访问。
Introduction
真核细胞中不断遇到各种需要快速适应性细胞反应有害的生理和环境应激条件。细胞应激反应涉及抗生存和促存活作用因子之间的精确平衡。破坏这种平衡可以有不可逆的后果导致人类疾病如癌症和神经退行性疾病的发展。在应激反应的第一步骤中，细胞激活，涉及改变基因表达在mRNA翻译水平的协调控制促存活途径。
在真核生物mRNA的翻译是涉及翻译起始因子（EIFS），特定的RNA结合蛋白（其中，RBD）和RNA分子1之间协调的相互作用复杂的细胞过程。 mRNA翻译被分成三个不同的阶段：起始，延伸和终止。虽然所有三个阶段都受到REGUlatory机制，平移控制机制，针对翻译的大多是起始阶段，这足以构成蛋白质的合成2的限速步骤。
翻译起始是一个高度有序的过程，开始于eIF2a.GTP.Met-tRNA的形成我遇到了三元复合物及其随后的结合到40S核糖体亚单位，从而导致预起始复合物的形成。接下来的步骤是将起始前复合物的mRNA，它涉及到翻译起始因子如eIF4F和eIF3的活性的募集。由此形成的48S起始前复合物经受特定的构象变化，使此机器开始扫描mRNA的5'-非翻译区，直到它识别起始密码子八月大部分的翻译起始因子，然后释放，60S亚基招募形成80S核糖体复合胜任翻译，一吨点蛋白质合成开始（ 图1），其中。多个80S monosome可翻译的mRNA相同的时间产生所谓的多聚核糖体（或者多聚核糖体）。多聚核糖体上的mRNA的密度反映了起始，延长和终止率，因而是一种特殊的转录物的可译性的度量。然而，多核糖更新主要用于在启动步骤，以评估变化的mRNA的翻译。在这里，我们使用了蛋白酶体抑制剂作为翻译起始抑制剂。癌细胞与这种药物治疗引起应激反应特点是激活的磷酸化翻译起始因子eIF2a 3名为HRI应力激酶。 eIF2a的磷酸化是重要的事件，导致翻译起始的在哺乳动物细胞中4的抑制作用1。
Protocol
该协议遵循的指导方针 经伐的伦理审查委员会。 1，制备细胞培养和脑操作哺乳动物和果蝇细胞生长的HeLa宫颈癌细胞和施奈德果蝇胚胎细胞所推荐的美国典型培养物保藏中心。与细胞在低通道工作。 板单元，以达到80％汇合的实验当天。为获得最佳结果，用〜12×10 6个细胞的每个实验条件。在进行提取物为多核糖体分析?…
Representative Results
如前面提到的，多核糖体轮廓允许翻译起始的胁迫条件下的变化的分析， 图1是如前面所述是一个涉及翻译起始复合物的有序组装多步骤的过程翻译起始的简化视图。在正常生长条件下，翻译起始复合物被转化为多核糖体，其检测由多核糖更新证实为一个活跃的翻译起始（ 图2;未处理）。根据应力条件然而，翻译起始被阻断导致80S monosomes积累和减少的多核糖体的峰（ <stro…
Discussion
在蔗糖梯度上的多核糖体轮廓分析允许翻译起始的测量通过分析多核糖体从细胞或组织9,11-14孤立的密度。这种技术是最好的（如果不是唯一的）方法来衡量翻译起始体内 。它是用来监视在细胞周期15生长的细胞的平移状态，并评估各种类型的应力，包括病毒感染，缺氧13,16，辐射17，和化学药物治疗中使用的18的作用，对翻译起始。然而，尽管这种?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
PA是奖学金“皮埃尔·杜兰德”的拉瓦尔大学医学院收件人。这项工作是支持由加拿大自然科学和工程研究理事会（MOP-CG095386）马币的多核糖体分馏是通过加拿大创新基金会赠款（MOP-GF091050）所收购，以RMR M拥有一个新的CIHR研究员薪水奖。
我们感谢博士。 E. Khandjian，一Gallouzi，S.迪 – 马可和A. Cammas的有益建议。
Materials
| Cells | ||||
| HeLa cervical cancer cells | American Type Culture Collection (Manassas, VA; ATCC) | CCL-2 | ||
| Schneider Drosophila embryonic cells | American Type Culture Collection (Manassas, VA; ATCC) | CRL-1963 | ||
| Culture medium and Supplements | ||||
| Schneider’s Drosophila Medium | Sigma-Aldrich | SO146-500ml | ||
| DMEM | Life technologies | 11995-073 | ||
| FBS | Fisher Scientist Scientist | SH30396-03 | ||
| penicillin/streptomycin | Life technologies | 15140122 | ||
| Sucrose solutions | ||||
| D-Sucrose | Fisher Scientist | BP220-212 | ||
| Glycerol | Sigma-Aldrich | 49767 | ||
| Blue Bromophenol | Fisher Scientist | B3925 | ||
| Lysis buffer | ||||
| Tris Hydrochloride | Fisher Scientist | BP153-500 | ||
| MgCl2 | Sigma-Aldrich | M2670-100G | ||
| NaCl | Tekniscience | 3624-05 | ||
| DTT | Sigma-Aldrich | D 9779 | ||
| Nonidet P40 (Igepal CA-630 ) | MJS Biolynx | 19628 | ||
| SDS | Tekniscience | 4095-02 | ||
| RNase inhibitor (RnaseOUT Recombinant Ribonuclease Inhibitor) | Life technologies | 10777-019 | ||
| Antiproteases (complete, mini, EDTA free) | Roche | 11,836,170,001 | ||
| RNA Extraction | ||||
| Proteinase K | Life technologies | AM2542 | ||
| Phenol: Chloroforme | Fisher Scientist | BP1754I-400 | ||
| Chloroforme | Fisher Scientist | C298-500 | ||
| Glycogen | Life technologies | 10814-010 | ||
| Isopropanol | Acros organics | 327270010 | ||
| Antibodies | ||||
| anti-FMRP antibody | Fournier et al., Cancer Cell International, 2010 | |||
| anti-Ribosomal Protein L28 antibody | Santa Cruz Biotechnology, Inc. | SC-50362 | ||
| Others | ||||
| Proteasome inhibitor : Bortezomib | LC Laboratories | B-1408 | ||
| DEPC (Diethylpyrocarbonate) | Sigma-Aldrich | D5758-25ml | ||
| RNaseZAP Solution | Life technologies | AM9780 | ||
| Materials | ||||
| T25 cell culture flask | Corning | 430639 | ||
| 1cc U100 Insulin Syringe 28 G1/2 | Fisher Scientist | 148291B | ||
| Tube ultra-centrifugation, PA, 12ml | Fisher Scientist | FSSP9763205 | ||
| Isco Model 160 gradient former | Teledyne Isco, Lincoln, NE, USA | |||
| Ultracentrifuge Sorvall OTD Combi | ||||
| Thermo Scientific Sorvall Rotor TH-641 | Thermo scientific | 54295 | ||
| Automated Density Fractionation System | Teledyne Isco, Lincoln, NE, USA | 67-9000-177 | ||
| Isco UA-6 UV-vis detector | Teledyne Isco, Lincoln, NE, USA | |||
| NanoDrop 2000 UV-Vis Spectrophotometer | Thermo scientific | |||
| Ultracentrifuge C 5415 | Eppendorf | |||
| Optical Microscope | Olympus CK2 | |||
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Citazione di questo articolo
Coudert, L., Adjibade, P., Mazroui, R. Analysis of Translation Initiation During Stress Conditions by Polysome Profiling. J. Vis. Exp. (87), e51164, doi:10.3791/51164 (2014).