应激条件下通过多聚核糖体分析检测翻译起始

摘要

本文介绍了一种用于分析真核细胞在应激条件下mRNA翻译起始变化的方法。该方法基于正在翻译的核糖体与非翻译核糖体在蔗糖梯度中沉降速度的差异进行分离。

摘要

mRNA翻译的精确调控对于真核细胞的稳态至关重要，尤其是在应对生理和病理应激时。该调控程序的异常可能导致受损细胞的异常增殖——这是癌症发展的标志，或导致细胞过早死亡，如在神经退行性疾病中所见。目前关于翻译调控分子机制的许多认识均来自利用密度梯度分离系统进行的多聚核糖体（polysome）分析。该技术依赖于将细胞质提取物在蔗糖线性梯度介质中进行超速离心。离心完成后，可对对应于不同翻译中核糖体群体的区带进行分级收集和定量，从而获得多聚核糖体图谱（polysome profile）。多聚核糖体图谱的变化反映了在多种应激条件下翻译起始过程发生的变化或缺陷。该技术还可用于评估特定蛋白质在翻译起始中的作用，并测量特定mRNA的翻译活性。本文中，我们描述了用于构建多聚核糖体图谱的实验方案，以评估真核细胞和组织在正常或应激生长条件下的翻译起始情况。

引言

真核细胞 constantly 遇到多种有害的生理和环境应激条件，需要迅速产生适应性细胞反应。细胞应激反应涉及促生存与抗生存作用因子之间的精确平衡。打破这种平衡可能导致不可逆的后果，引发癌症和神经退行性疾病等人类病理学疾病的发生。在应激反应的第一阶段，细胞会激活涉及在 mRNA 翻译水平上协调调控基因表达变化的促生存通路。

真核生物中的 mRNA 翻译是一个复杂的细胞过程，涉及翻译起始因子（eIFs）、特定的 RNA 结合蛋白（RBDs）与 RNA 分子之间的协调相互作用¹。mRNA 翻译分为三个明确的阶段：起始、延伸和终止。尽管这三个阶段均受到调控机制的影响，但翻译调控机制主要靶向翻译的起始阶段，因此该阶段构成了蛋白质合成的限速步骤²。

翻译起始是一个高度有序的过程，始于eIF2α·GTP·Met-tRNA_i^Met三元复合物的形成，随后该复合物与40S核糖体亚基结合，从而形成翻译前起始复合物。下一步是将前起始复合物招募至mRNA，此过程涉及翻译起始因子如eIF4F和eIF3的参与。由此形成的48S前起始复合物经历特定的构象变化，使该复合物能够沿mRNA的5'非翻译区开始扫描，直至识别起始密码子AUG。随后，大部分翻译起始因子被释放，60S亚基被招募，形成具有翻译能力的80S核糖体复合物，此时蛋白质合成开始（图1）。在同一时间，多个80S单体核糖体可同时翻译同一条mRNA，形成所谓的多聚核糖体（或多聚核糖体）。mRNA上多聚核糖体的密度反映了翻译的起始、延伸和终止速率，因此可作为特定转录本翻译效率的衡量指标。然而，多聚核糖体图谱主要用于评估mRNA翻译在起始阶段的变化。在本实验中，我们使用了一种蛋白酶体抑制剂作为翻译起始抑制剂。用该药物处理癌细胞会诱导一种应激反应，其特征是应激激酶HRI被激活，进而磷酸化翻译起始因子eIF2α³。eIF2α的磷酸化是导致哺乳动物细胞中翻译起始受到抑制的主要事件之一⁴。

方案

The protocol follows the guidelines approved by Laval's Ethical Review Board.

1. Preparation of Cell Cultures and Brain Manipulation

1. Mammalian and Drosophila Cells
   1. Grow HeLa cervical cancer cells and Schneider Drosophila embryonic cells as recommended by the American Type Culture Collection. Work with cells at a low passage.
   2. Plate cells in order to reach 80% confluence the day of the experiment. For best results, use ∼12 x 10⁶ of cells for each experimental condition. Before making extracts for polysome analysis, stimulate translation by adding fresh complete medium for at least 90 min.
2. Brain isolation: Isolate brains from mice aged between 9 and 16 days after birth. Euthanasia involves CO₂ inhalation after anesthesia and cervical dislocation. Following sacrifice, isolate the whole brain and either place it at -80 °C or directly transfer it into cold PBS 1X for immediate use.

2. Preparation of the Density Gradient Fractionation System

1. Wash the tubing system with 0.1% SDS for 5 min.
2. Wash the tubing system with 70% Ethanol for 5 min, then with RNaseZAP Solution for 5 min before washing it with DEPC water.
3. Pump air in order to dry the tubing system of the apparatus.

3. Preparation of the Sucrose Gradients

1. Make 15% and 55% sucrose solutions in 20 mM Tris-HCl pH 7.4, 1.25 mM MgCl₂, 150 mM NaCl, and 1 mM DTT.
2. Generate the linear 15-55% sucrose gradient using an Isco Model 160 gradient former, as described by the manufacturer's instructions. However, it is important to control well the TRIS peristaltic pump speed in order to avoid creation of bubbles or turbulences in the gradients. Keep the gradients at 4 °C until use.
3. During the time when the gradient maker program is running, cool the ultracentrifuge to 4 °C.

4. Preparation of Cell and Mouse Brain Extracts

1. Preparation of Cell Extracts
   1. Place plate(s) on ice and wash cells 3x with cold PBS 1X.
   2. Harvest cells (∼12 x 10⁶) in 1 ml lysis buffer (20 mM Tris-HCl at pH 7.4, 1.25 mM MgCl₂, 150 mM NaCl, 1 mM DTT, 1% Nonidet P40, 5 U/ml RNase inhibitor, supplemented with complete mini EDTA-free protease inhibitor cocktail tablets), transfer them to an Eppendorf tube, and mix well by passing them 15x through a 1cc U100 Insulin Syringe 28 G 1/2. Let the cell lysate rest on ice for 15 min.
   3. Put few drops of cell lysate onto a slide to assess cellular lysis by observing at a phase contrast microscope using a 10X objective. Only nuclei should be visible and no cell membranes should be visible attesting for cell lysis. As a control, observe unlysed cells.
   4. Clarify the cell lysate by centrifugation at 11,000 x g for 20 min at 4 °C and keep the soluble lysate containing the polyribosomes.
2. Preparation of Mouse Brain Extracts
   1. Homogenize the whole isolated brain (∼500 mg) by 10 strokes in an ice-cold Dounce homogenizer with 2 ml of lysis buffer and clarify the homogenate by centrifugation at 11,000 x g for 15 min at 4 °C.
   2. Load the resulting supernatant onto a cushion of 50% sucrose and proceed with sedimentation for 2 hr at 200,000 x g using an ultracentrifuge rotor TH-641 at 4 °C.
   3. Resuspend the translucent pellet containing the polyribosomes in 1 ml of lysis buffer and mix well by pipetting up and down. Let the suspension rest on the ice for 30 min before loading onto gradients.

5. Loading the Extracts onto Sucrose Gradients and Ultracentrifugation

1. Measure the concentration of RNA present in the cytoplasmic extract using a spectrophotometer. Carefully and slowly load ∼20 OD₂₆₀ units of the extract onto the 15% to 55% sucrose gradient. Make sure that there is 2-3 mm of available space at the surface of tube to avoid overflowing the tubes during centrifugation.
2. Centrifuge the gradients using an ultra-centrifuge for 2 hr 30 min at 230,000 x g using an ultracentrifuge rotor TH-641 at 4 °C. When centrifugation ends, remove the tubes and place them on ice carefully not to disturb the gradients.

6. Fractionation of Cytoplasmic Extracts for Polysomes Profiling

1. Place sucrose gradients on Automated Density Fractionation System and proceed with automated fractionation and collection of fractions (0.5 ml each), as described by the manufacturer.
2. Collect each fraction (∼500 µl) into individual Eppendorf tubes with continuous monitoring of absorbance at 254 nm. In parallel of the gradient fractionation operation, the polyribosomal profile will be editing on the chart paper. Alternatively, use a data acquisition unit attached to the chart recorder to have an electronic acquisition of the polysome profile.
3. At the end of each run, transfer collected Eppendorfs on dry ice. At this time, either store collected fractions at -80 °C or directly precipitate protein-RNA complexes as follows.

7. Protein Extraction and Analysis

1. To each collected fraction of sucrose gradient, add 2 volumes of 100% cold ethanol and let RNA-protein complexes precipitate at -20 °C overnight.
2. Centrifuge each RNA-protein precipitate at 11,000 x g for 20 min at 4 °C then wash with 70% ethanol. Dry and resuspend the precipitate in SDS-PAGE sample buffer before proceeding with Western blot using specific antibodies (see Figure 2).

8. RNA Extraction and Analysis

1. Precipitate RNA-protein complexes as in section 7.1. Resuspend each RNA-protein precipitate in the lysis buffer containing 0.1% SDS. Digest the protein component of the precipitate with 2 mg/ml Proteinase K for 30 min in a 55 °C water bath.
2. Extract RNA components of the precipitate by adding 1 volume of "phenol: chloroform", 2 volumes of chloroform, and 0.1 volume of 2 M NaOAc pH 4 and centrifuging at 10,000 x g at 4 °C for 20 min. Precipitate RNA from the resulting aqueous phase of each sample overnight at -20 °C by adding 1 volume of isopropanol and 0.2 µg/µl of glycogen. Spin the precipitate for 1 hr at 10,000 x g at 4 °C. Wash the RNA pellet with 70% cold ethanol.
3. Resuspend the RNA pellet into a small volume of RNAse free water. Assess the quantity and quality of RNA using the spectrophotometer.

结果

如前所述，多聚核糖体图谱可用于分析在应激条件下翻译起始的变化。图1为翻译起始过程的简化示意图，如前所述，该过程为多步骤过程，涉及翻译起始复合物的有序组装。在正常生长条件下，翻译起始复合物转化为多聚核糖体，通过多聚核糖体图谱检测到这些多聚核糖体，表明翻译起始处于活跃状态（图2；未处理组）。然而，在应激条件下，翻译起始受到阻断，导致80S单体核糖体积累，同时多聚核糖体峰减少（图2；蛋白酶体抑制剂处理组）。

讨论

蔗糖梯度中的多聚核糖体图谱分析可通过检测从细胞或组织中分离的多聚核糖体的密度来评估翻译起始效率^9,11-14。该技术是目前测量体内（in vivo）翻译起始的最优（甚至可能是唯一）方法。它被用于监测细胞周期过程中增殖细胞的翻译状态¹⁵，并评估多种应激因素（包括病毒感染、缺氧^13,16、辐射¹⁷以及治疗中使用的化学药物¹⁸）对翻译起始的影响。然而，尽管该技术在评估细胞培养物及特定组织（如脑和肝）的翻译起始方面效果良好，但其是否可用于比较正常组织与病变组织及肿瘤之间的翻译起始水平，仍有待确定。

在影响翻译延伸或终止的条件下，多核糖体图谱也可能发生变化。在这些情况下，翻译延伸速率的抑制将导致多核糖体峰增加，而终止过程的抑制则会导致相对于对照组出现更大的多核糖体。相反，翻译起始抑制剂会阻止多核糖体的重新形成，因为活性翻译起始复合物的形成被阻断。这在多核糖体图谱中表现为多核糖体峰减少，同时伴随80S单体核糖体的增加。由于翻译抑制剂使翻译起始复合物停滞，理论上40S和...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
细胞
HeLa 宫颈癌细胞	美国模式培养物集存库（弗吉尼亚州马纳萨斯；ATCC）	CCL-2
Schneider Drosophila 胚胎细胞	美国模式培养物集存库（弗吉尼亚州马纳萨斯；ATCC）	CRL-1963
培养基与添加剂
Schneider’s Drosophila 培养基	Sigma-Aldrich	SO146-500ml
DMEM	Life technologies	11995-073
FBS	Fisher Scientist	SH30396-03
青霉素/链霉素	Life technologies	15140122
蔗糖溶液
D-蔗糖	Fisher Scientist	BP220-212
甘油	Sigma-Aldrich	49767
溴酚蓝	Fisher Scientist	B3925
裂解缓冲液
Tris HCl	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 抑制剂（RnaseOUT 重组核糖核酸酶抑制剂）	Life technologies	10777-019
抗蛋白酶（complete, mini, EDTA free）	Roche	11,836,170,001
RNA 提取
蛋白酶 K	Life technologies	AM2542
苯酚:氯仿	Fisher Scientist	BP1754I-400
氯仿	Fisher Scientist	C298-500
糖原	Life technologies	10814-010
异丙醇	Acros organics	327270010
抗体
抗 FMRP 抗体	Fournier et al., Cancer Cell International, 2010