快速 体内 真核细胞中翻译复合物的固定与分离

摘要

我们介绍一种利用甲醛交联在活酵母和哺乳动物细胞中快速稳定翻译（蛋白质生物合成）复合物的技术。该方法能够解析瞬时中间产物以及动态的RNA:蛋白质相互作用。交联后的复合物可用于多种下游应用，例如基于深度测序的分析方法、显微镜观察和质谱分析。

摘要

涉及信使RNA（mRNA）快速重分布及mRNA翻译改变的快速响应，与细胞正在进行的稳态调节密切相关。这些调节对于真核细胞在营养和盐度波动、温度变化以及各种化学和辐射胁迫条件下的存活能力和“损伤控制”至关重要。由于RNA水平响应具有高度动态性，且许多RNA:RNA和RNA:蛋白质中间体不稳定，因此仅通过有限的几种方法才能获得有意义的细胞质RNA状态快照。全转录组范围、基于RNA测序的核糖体谱分析（ribosome profiling）类实验是研究翻译调控最具信息量的数据来源之一。然而，若缺乏对RNA及RNA:蛋白质中间体的统一稳定化处理，则可能导致不同的偏差，尤其是在快速响应的细胞通路中。本文提供了一种适用于不同通透性真核细胞的快速固定详细方案，以辅助稳定RNA及RNA:蛋白质中间体。我们进一步提供了基于核糖体及多（核糖）体组分共沉降方法分离稳定化RNA:蛋白质复合物的示例。所分离的稳定化材料可后续用于核糖体谱分析类实验，例如翻译复合物谱分析（Translation Complex Profile sequencing, TCP-seq）及其衍生技术。目前，TCP-seq类方法的通用性已在多种生物体和细胞类型的应用中得到验证。由于交联易于逆转，这些稳定化的复合物还可进一步进行亲和纯化，并通过电子显微镜成像，或分离至不同的多（核糖）体组分后进行RNA测序。因此，基于瞬时冷冻和甲醛固定的处理方法，结合基于沉降或其他类型的RNA:蛋白质复合物富集技术，特别适用于在活细胞中深入探究快速RNA:蛋白质复合物动态的精细机制。

引言

生物体在其生命周期中会经历动态的细胞内和细胞外变化，需要快速响应以维持稳态并确保生存。为了适应环境变化，真核细胞通过基因表达调控来调节其代谢过程。via 基因表达的调控可发生在转录和/或翻译阶段，其中翻译水平的响应通常更为迅速^1,2,3,4。例如，翻译水平的变化通常在应激发生后1至30分钟内出现，而转录水平的改变则在应激暴露数小时后才发生^3,4,5。由于信使RNA（mRNA）分子在细胞质中持续存在，翻译产物的改变能够更快实现。相反，在转录水平上，必须合成新的mRNA分子，并且在真核生物中还需经过加工并从细胞核输出，导致响应时间显著延迟^2,4,6,7,8。

急性应激下的翻译反应通常表现为整体翻译产物的减少，同时选择性上调对细胞存活至关重要的蛋白质^1,3,4,9。由于翻译过程消耗大量能量，降低蛋白质合成输出被认为至关重要^3,7。为实现选择性抑制与上调，翻译反应依赖于一系列复杂的调控机制。这些调控可作用于翻译的所有阶段：包括多肽生物合成的起始、延伸、终止以及核糖体的再循环^10,11,12,13，但主要在起始阶段表现最为显著^5,7,9,10,13。在起始阶段，小核糖体亚基（SSU）在真核起始因子（eIFs）的协助下结合到mRNA的5'非翻译区（UTR），并沿其扫描，直至识别起始密码子^{2,5,6,8,11,12,13}。调控机制通常靶向影响结合、扫描及起始密码子识别的eIFs。例如，起始因子eIF2是一种关键的翻译因子，可协助将起始Met-tRNA_i^Met招募至SSU，在应激条件下常成为真核生物中的调控靶点^4,6,11。在酵母中，营养缺乏和渗透胁迫可诱导该因子的磷酸化^1,4,11,14,15；而在哺乳动物细胞中，氨基酸饥饿、内质网（ER）应激、紫外线应激、病毒感染以及氧水平变化均可触发这一反应^8,9,11。在哺乳动物细胞对缺氧的反应中，特定mRNA翻译的快速上调尤为明显，表现为全局性的快速翻译抑制以及缺氧诱导因子（HIFs）生物合成的选择性上调。HIFs作为转录因子，进一步在DNA转录水平引发长期的细胞重编程^8,9,16。类似反应也见于热应激下的酵母，表现为热休克蛋白（HSPs）的快速翻译表达，随后出现延迟的转录水平响应^17,18。除营养缺乏和热休克外，酵母在不同氧浓度^8,19、盐度⁵、磷酸盐、硫^20,21和氮^22,23水平下也已观察到翻译反应。该研究对酵母在烘焙和发酵等工业应用具有广泛意义^24,25。翻译反应还有助于深入理解神经退行性疾病和心脏病等疾病，这些疾病常伴随氧化应激等细胞内应激状态。总体而言，翻译反应是真核生物基因表达调控的重要组成部分，有助于快速适应多种应激条件。

为了研究翻译响应，需要采用能够提供翻译景观最小失真快照的方法。多聚核糖体分析是一种用于研究mRNA翻译的经典方法，涉及分离mRNA的多聚（核糖）体组分 通过 蔗糖梯度超速离心^26,27该方法可用于研究单个mRNA的翻译水平（通过逆转录聚合酶链反应等检测方法，RT-PCR）²⁶），或结合高通量技术（微阵列或RNA-seq）进行全局分析^28,29）。更进一步的方法是核糖体谱分析，该方法能够在全基因组范围内研究延伸中的核糖体在mRNA分子上的位置，同时推断翻译效率在转录组中的分布以及主要和替代起始位点的利用情况^30,31核糖体谱分析涉及分离并测序由核糖体结合所保护的mRNA片段。核糖体谱分析已在多种条件下揭示了翻译动态的大量信息，包括缺氧应激、热休克和氧化应激。^31,32该技术已适用于多种来源材料，包括酵母和哺乳动物细胞。

尽管多聚核糖体分析和核糖体谱型分析在拓展翻译研究能力方面发挥了重要作用，但翻译过程包含多种翻译中间体和复合物，这些方法难以捕获。^11,13. 另一个局限性源于无法研究快速响应类型，因为翻译复合物要么被稳定化 在体 通过添加特定的翻译抑制剂（抗生素），导致某些核糖体分布的人工假象，或 离体 通过细胞裂解特异性（抗生素）或非特异性（高盐或镁离子）地剥夺寿命较短或稳定性较差的中间产物^33,34,35.

甲醛被广泛用于交联核酸与蛋白质，例如在染色质免疫沉淀（ChIP）和交联免疫沉淀（CLIP）研究中。由于其分子小且细胞通透性优异，可实现快速的体内作用³⁶。基于甲醛的快速交联特性，核糖体谱分析技术已扩展为翻译复合物谱测序（TCP-seq）^{10,36,37,38,39,40}。TCP-seq 最初在酵母中建立，能够捕获所有翻译中间体，包括扫描或翻译终止后的SSU复合物以及多种核糖体构象^37,38,41,42。该方法已在多项研究中得到应用^{10,38,39,41,42}，其中一些研究结合使用翻译抑制剂与甲醛交联，以促进翻译过程的停滞。该技术的一个进一步改进版本——选择性TCP-seq（selective TCP-seq）³⁹，最近被用于引入对交联复合物的免疫纯化，从而拓展了TCP-seq的应用范围。甲醛交联具有快速、高效且可逆的特性，使这些方法适用于研究瞬时的mRNA:翻译复合物相互作用，特别是在高度动态的翻译水平响应通路中的应用。

本文详细阐述了为实现翻译复合物的全面稳定与分离而进行的体内甲醛交联操作流程。我们分别提供了适用于酵母和哺乳动物细胞的精细化实验方案（图1）。此外，我们还概述了交联稳定化材料的后续应用示例（图1），包括利用免疫印迹（Western blotting）检测共纯化的蛋白质因子、免疫辅助纯化（或称“免疫沉淀”；IP）以富集含有特定目标因子的翻译复合物、电镜分析以及RNA测序。

图1示意图展示了典型实验装置的概览。 主要步骤 体内 翻译复合物的甲醛交联稳定化流程以流程图形式展示，并补充了关键所需仪器的相关信息。文中概述了交联产物可能的下游应用，包括一些已成功使用但本方案未直接涵盖的方法实例，如RNA的SPRI磁珠纯化、RNA测序以及质谱分析。 请点击此处查看此图的放大版本。

方案

1. Yeast cell protocol

1. Yeast cell culture and fixation
   NOTE: Cell fixation and harvesting are adapted from^10,38 with modifications.
   1. Set up 1 L yeast cell culture (wild-type (WT) BY4741 are given as an example) in an orbital shaker with the starting optical density of no more than 0.05 AU at 600 nm (OD₆₀₀) in suitable media (1% w/v of yeast extract, 2% w/v of peptone, 2% w/v of dextrose (glucose), 40 mg/L of adenine sulfate (YPD) used as an example) under the desired conditions (30 °C used in this experiment).
   2. Set up a preparative centrifuge with compatible rotor and centrifuge bottles for pelleting the liquid suspension culture of yeast cells. For glucose starvation experiments, pellet the cells once the optical density of 0.6-0.8 AU at 600 nm (OD₆₀₀) is reached, using a brief centrifugation at 30 °C, 5,000 x g for 1 min.
      NOTE: Keep record of the OD of the growing cells and let the cells grow until the OD₆₀₀ reaches 0.6-0.8 AU, if the exponential growth phase is of an interest.
   3. Resuspend the pellet immediately in warm (30 °C) YP media containing no or low (0.25% w/v) added glucose and incubate the culture for a further 10 min at 30 °C in an orbital shaker-incubator.
      NOTE: Media composition might affect subsequent crosslinking efficiency. This protocol was tested using YPD only. When performing starvation experiments, adhering to the timing and minimizing the delays between procedures is critical.
   4. Once the cells are ready, set up an ice box inside the fume hood with a beaker containing 250 g of clean crushed water ice. Ensure 25 mL stripettes and freshly purchased methanol-stabilized 37% w/v formaldehyde solution are accessible inside the hood. Pour the 1 L culture into the beaker containing 25% w/v of crushed water ice.
      NOTE: Keep the cells on ice throughout all subsequent operations until the cells are frozen, unless indicated otherwise.
   5. Add 75 mL of 37% w/v of formaldehyde solution to a final concentration of 2.2% w/v and intensely stir the mixture until the ice melts.
   6. Once the ice is melted, set up a timer for 10 min.
      NOTE: Adhere to the recommended timings and temperature regimen to attain reproducible fixation results.
   7. After incubating for 10 min, transfer the culture into the precooled centrifuge bottles and pellet the cells by centrifugation at 4 °C, 5,000 x g for 5 min. While this spin is on, precool a 50 mL tube and keep freshly prepared buffer A (containing glycine to neutralize any remaining formaldehyde) on ice.
      NOTE: Refer to the supplied table for the exact buffer compositions.
   8. After centrifuging, place the centrifuge tubes onto ice with the pellet side in contact with the ice. Bring the tubes into the fume hood and discard the supernatant into a formaldehyde waste container.
   9. Resuspend the cell pellet from all tubes in 20 mL of buffer A using a 25 mL stripette and transferring to a 50 mL tube.
      NOTE: This wash is critical for avoiding irreproducible crosslinking and the buffer addition must not exceed 20 min of time from the cells' harvest.
   10. Make the volume up to 40 mL with buffer A and collect the washed cells by centrifugation at 4 °C, 5,000 x g for 5 min.
   11. Discard the supernatant and resuspend the cell pellet in 40 mL of buffer A1, which is buffer A not containing glycine, to remove any glycine contamination.
   12. Pellet cells again by centrifugation at 4 °C, 5,000 x g for 5 min.
   13. Repeat the washes with buffer A1 one more time. Discard the supernatant and place the cell pellet on ice. Weigh the tube with the pellet (wet cell mass should be ~1 g per 1 L of the cell culture).
2. Yeast cell disruption and cytosol collection
   1. Fill a polystyrene foam box lined with aluminum foil with liquid nitrogen to a depth of approximately 3 cm. Place a 50 mL tube upright in the box.
   2. Resuspend the pellet (~1 g wet cell mass) in 550 µL of buffer A2 by pipetting and vortexing for 10 s. Add 10 µL of 40 U/µL of RNase inhibitor and vortex again for 10 s.
      CAUTION: Wear appropriate protective equipment, such as thermally insulated gloves, when handling liquid nitrogen. Ensure that any container used to hold liquid nitrogen does not leak, and that the tube rack inside will not float up or fall on its side. Work in a well-ventilated area to avoid oxygen depletion.
   3. Using a 1 mL pipette, drip the cell suspension into the 50 mL tube containing the liquid nitrogen.
      NOTE: The dripping must be performed slowly and carefully to avoid aggregation of the droplets. Ensure the droplets freeze before introducing new droplets.
   4. Transfer the 50 mL tube with the frozen cell suspension droplets to room temperature and wait until the liquid nitrogen evaporates completely. Seal the tube with its cap and store the cell pellets at -80 °C or immediately proceed further.
      CAUTION: Ensure that the liquid nitrogen is completely evaporated before sealing the tube. Leftover liquid nitrogen in a sealed tube can cause a hazardous pressure build-up.
   5. To prepare for the next step, precool 1.5 mL nuclease-free tubes and 10 mL stainless steel grinding jars on dry ice.
   6. Transfer the frozen cell suspension droplets into the jars using a clean, sterile spatula.
      CAUTION: Ensure that the grinding jars are tightly sealed.
   7. Submerge the grinding jars into the liquid nitrogen for 1 min ensuring the liquid phase remains below the junction. Set up a cryo mixer mill at 27 Hz for agitation for 1 min.
      NOTE: Always balance the grinding canister with another one of the same model even if the sample requires only one canister for processing.
   8. Agitate the sealed grinding jars at 27 Hz for 1 min in the mixer mill.
   9. Re-cool the grinding jars in liquid nitrogen as before and shake at 27 Hz for 1 min further in the mixer mill.
   10. Transfer the jars to the ice box containing dry ice along with the 1.5 mL nuclease-free tubes. Using a small steel spatula, transfer the resultant powdered sample into the tubes in ~100 mg aliquots, and store the tubes at -80 °C.
       ​NOTE: It is recommended to use ~600 mg of the sample per experiment comprising polysome sedimentation profile analysis, separation of the cytosol into translated and non-translated fractions, and further separation of the translated fraction into SSU, ribosome, and disome fractions upon RNase digestion.
3. Separation of the fixed (poly)ribosomal complexes from the non-translated fractions of the cytosol
   NOTE: The procedure established earlier^10,38 is generally followed to enrich translated RNA based on its co-sedimentation with (poly)ribosomes. A more refined approach to separating the translated and non-translated cytosol fractions is introduced here, eliminating the need to precipitate and subsequently re-solubilize the material.
   1. Prepare 2.5 mL linear 10%-20% w/v sucrose gradients with buffer B using the freeze-thaw method⁴³ in thin wall ultracentrifuge tubes (5 mL, 13 x 51 mm).
      NOTE: The freeze-thaw method is performed by the sequential addition and freezing of buffered sucrose layers with linearly regressing concentrations on top of each other. See Supplementary Table 1 for details.
   2. To create a discontinuous 50% w/v sucrose cushion, upon the linear gradients thawing and stabilizing, slowly dispense 0.5 mL of 50% sucrose in buffer B directly onto the bottom of the tubes using a 1 mL syringe attached to 19 G x 1.5" needle or a glass capillary of similar/suitable dimensions. Before dispensing, carefully and slowly drive the tip of the needle or capillary from the top to bottom of the pre-formed sucrose gradients, avoiding any disturbance, until it reaches the tube bottom.
      NOTE: See Supplementary Table 1 for instructions on preparation of buffer B.
   3. Carefully balance the gradients by removing the top portions or layering more 10 w/v of sucrose in buffer B and keep them ice-cold or at 4 °C.
      NOTE: The discontinuous gradient with the bottom 50% sucrose layer is needed to collect material with higher sedimentation rate without precipitating it on the tube wall.
   4. Thaw ~100 mg of the frozen cell powdered sample at room temperature and immediately place on ice. Mix in 150 µL of buffer A2 by pipetting, add RNase inhibitor to 1 U/µL and mix by vortexing (avoid excessive foaming and mixing with the gaseous phase) for 10 s.
      NOTE: Continue all operations while keeping the material on ice, unless otherwise indicated.
   5. Pellet the cell debris by centrifuging the tubes at 4 °C, 13,000 x g for 5 min and recover the clarified supernatant (~150 µL) in a new 1.5 mL low protein binding tube.
   6. Load the resultant clarified mixture onto the discontinuous sucrose gradient tubes from step 1.3.3 and carefully balance them.
   7. Ultracentrifuge the tubes in a medium volume swing-bucket rotor at 4 °C, with average g-force 287,980 x g (k-factor 49) for 1 h 30 min.
      NOTE: These conditions have been pre-optimized (using post-ultracentrifugation gradient UV absorbance trace analysis) to retain the free (non-(poly)ribosomal) SSUs and LSUs (large ribosomal subunit) in the top (10%-20% sucrose) portion of the gradient while concentrating the (poly)ribosomal fraction in the bottom (50%) sucrose cushion without pelleting the material.
   8. Use a new sterile 1 mL syringe equipped with a 19 G x 1.5" needle to collect the translated cytosol fraction. Place the 5 mL gradient on a stable rack ensuring the bottom of the tube is visible.
   9. From the top of the tube, stick the needle straight into the bottom of the gradient (without puncturing the tube) and gently, without creating any bubbles, draw exactly 0.5 mL of the bottom solution containing the translated RNA pool.
      NOTE: Ensure this step is performed in a cold room and the tube is held firmly. It is recommended to draw the entire 0.5 mL in a single upstroke motion to avoid disturbance of the gradient.
   10. Confirm the (poly)ribosomal presence and the depletion of the SSU, LSU and lighter fractions in the resultant mixture by absorbance readout of the sucrose gradient upon ultracentrifugation run.
   11. Concentrate the collected translated RNA pool from the previous step to 100 µL using ultrafiltration in a micro-concentration device with 10 kDa cut-off regenerated cellulose membrane.
       NOTE: Pre-wash the micro-concentration device's membrane with 0.5 mL of buffer 1 (see Figure 2a) and use spin conditions (g) recommended by the manufacturer.
   12. Further dilute the material from the previous step five times (add 400 µL) with buffer 1 and concentrate back to 200 µL, to allow for a smaller volume as well as partial removal of the sucrose.
       NOTE: It is recommended to store the resultant mixtures at -80 °C for up to 6 months and use as an input material for the 'total translated RNA' RNA-seq library construction, or the RNase digestion step of the TCP-seq library construction. The 'non-translated' cytosol fraction can be recovered from the top of the gradient using a similar procedure and stored at -80 °C.
4. RNase digestion of the fixed (poly)ribosomal complexes and separation of digested material into small ribosomal subunit (SSU), monoribosomal (ribosomes, RS), and diribosomal (disomes, DS) fractions
   NOTE: The procedure generally follows an approach described previously^10,38 but a modified gradient type, separation time, acceleration and RNase digestion conditions are employed, to achieve best resolution across all three isolated fractions.
   1. Prepare carefully balanced 12.5 mL linear 10%-40% w/v sucrose gradients made with buffer 1 in 13 mL thin wall polypropylene tubes, 14 x 89 mm, using the freeze-thaw method⁴³ as described in step 1.3.1 and note therein.
   2. Thaw at room temperature and immediately transfer the samples on ice or take the concentrated and sucrose-depleted translated cytosol fraction from step 1.3.12.
      NOTE: Continue all procedures on ice unless otherwise indicated.
   3. Digest the translated cytosol fraction by mixing in 4.5 U of E. coli RNase I per 1 OD₂₆₀ unit of the fraction for 30 min at 23 °C. Immediately add and mix in by pipetting the RNase inhibitor capable of inactivating RNase I to 0.25 U/µL to the mixture, to inactivate RNase I.
      NOTE: Use RNase inhibitor capable of inhibiting RNase I. Derive AU₂₆₀ by using AU₂₆₀ = (Absorbance at 260 nm standardized to optical density units equivalent to 1 cm optical path x volume of the lysate in µL) / 1,000.
   4. Immediately transfer the samples to ice.
      CAUTION: It is critical to adhere to the recommended conditions of digestion and carefully measure the amount of the added RNase I. The RNase I unit referred to here is defined as the amount of the enzyme required to produce 1 µg of acid-soluble material from mouse liver RNA in 30 min at 37 °C. RNase I batches may have undocumented variations in activity and may require experimentation to achieve optimal digestion conditions. If the enzyme stock is too concentrated, it is recommended to dilute it with buffer 1 to avoid pipetting very small volumes of the solution.
   5. Load the reaction mixtures onto the 10%-40% w/v sucrose gradients from step 1.4.1.
      NOTE: Use final volumes in the range of 150-300 µL per gradient. Each purification requires minimally two gradients. Use different input volumes of the material (lower AU₂₆₀, 10-11 AU₂₆₀, for DS and comparatively higher AU₂₆₀, 13-14 AU₂₆₀, for SSU or RS) to achieve optimal separation.
   6. Ultracentrifuge the tubes in a medium volume swing-bucket rotor at 4 °C with average g-force 178,305 x g (k-factor 143.9) for 3 h 30 min.
      CAUTION: If spare balance tubes are needed, equalize their mass and mass distribution with the sample-containing tubes. Use spare sucrose gradients overlaid with an amount of buffer equivalent to that of the sample overlay and not tubes with uniform sucrose concentration.
   7. Set up a gradient fractionator device at least 30 min before the ultracentrifugation spin completion, including filling in the 0.2 µm filtered heavy chase solution (e.g., 60% sucrose in deionized water as used here) into the displacement pump.
      NOTE: It is recommended to de-contaminate the lines and tubings of the fractionator using deionized water, followed by 1%-2% SDS solution in deionized water, deionized water, and finally 80% ethanol in deionized water solution before and after the runs.
   8. Adjust the absorbance readout baseline by first filling the system with deionized water and zeroing the optics as per the manufacturer's recommendations, and then compensating the baseline shift using a spare unloaded 14 x 89 mm sucrose gradient made with a buffer identical to the sample tubes (e.g., buffer 1).
      NOTE: Use the same displacement speed to make the adjustments as for the sample readout, such as 1.5 mL/min.
   9. Measure the displacement system dead volume by accurately counting time between the solution first entering the optical path of the detector and first appearing at the fraction collector output.
      NOTE: With the recommended speed of 1.5 mL/min, the fractionation can be performed at room temperature. It is recommended to immediately transfer the collected fractions on ice.
   10. Perform fractionation using live absorbance readout at 254 nm, 1.5 mL/min displacement speed, and in-line fraction detection based on the expected sedimentation position and absorbance profile of the samples. Use collector tube switching with a time delay corresponding to the dead volume as measured before.
   11. Isolate fractions corresponding to the positions and mobility of the SSU, RS, and DS complexes and collect them into new low protein binding 1.5 mL microcentrifuge tubes; immediately transfer the isolated fractions on ice and freeze if not processed further right away.
       NOTE: It is recommended to immediately flash-freeze the collected fractions in dry ice or liquid nitrogen and store at -80 °C or below for up to 6 months.
5. De-crosslinking of the ribosomal complexes and isolation of the RNA to construct RNA-seq libraries
   1. To de-block/reverse the crosslinks and isolate the RNA away from the associated proteins, transfer approximately half of the entire sucrose gradient fractions into new low nucleic acid binding nuclease-free polypropylene 1.5 mL microcentrifuge tubes (350 µL per tube) with lid safety/locking devices.
   2. Supplement the mixtures with 40 µL of 100% stop solution (10% SDS w/v and 100 mM EDTA), 4 µL of 1 M Tris-HCl pH 2 at 25 °C (to 10 mM), 1.6 µL of 2.5 M glycine (to 10 mM) and deionized nuclease-free water to obtain the final volume of 400 µL.
   3. Mix the contents of the tubes by pipetting and transfer the tubes at room temperature.
   4. Add equal volume of the acidic phenol:chloroform:isoamyl alcohol 125:24:1 (pH 4.0-5.0) mixture to each tube. Vigorously shake the mixtures for 2 min using a vortex mixer set to maximum speed.
      CAUTION: Phenol and chloroform are corrosive and toxic. Avoid physical contact with the liquids and work in a well-ventilated area or under a fume hood. Always use gloves, lab coat and protective goggles or a face shield when working with phenol or chloroform.
   5. Place the tubes in a thermoshaker and continuously shake at 65 °C, 1,400 rpm for 30 min.
   6. Facilitate phase aggregation by centrifuging the mixture at 12,000 x g for 10 min at room temperature.
   7. Collect the upper aqueous phases and transfer them into fresh low nucleic acid binding 1.5 mL tubes.
      NOTE: To avoid cross-contamination, do not attempt to recover the aqueous phases completely. A reasonable recovery volume is 300-350 µL.
   8. Supplement the collected aqueous phases with 0.1 volumes of 3 M sodium acetate (pH 5 at 25 °C), 20 µg of glycogen (using 5 µg/µL stock) and 2.5 volumes of absolute ethanol. Carefully mix the solutions by vortexing the tubes for 1 min.
   9. Precipitate the RNA by incubating the samples at -20 °C for at least 2 h (recommended overnight).
   10. Warm the tubes to room temperature and mix by vortexing.
       NOTE: Pre-warming of the tubes and subsequent centrifugation at room temperature (without forced chilling) help to reduce salt and phenol co-precipitation and carryover. These conditions should not result in material loss or inefficiency of RNA collection if performed as described and using sufficiently pure ethanol.
   11. Pellet the RNA precipitate by centrifuging the tubes at 12,000 x g for 30 min at room temperature.
   12. Discard the supernatant and wash the pellet twice with 80% v/v ethanol, collecting it each time by centrifugation at 12,000 x g for 10 min at room temperature.
   13. Dry the RNA pellets by opening the tube lids and placing the opened tubes in a dry-block heater set to 45 °C for 10 min. Dissolve the resultant dried pellet in 20 µL of 1x HE buffer.
   14. Estimate the resultant RNA concentration using UV absorbance spectrum measurement.
       NOTE: RNA fragment length and total amount can be further assessed using denaturing gel-electrophoresis, such as in an automated fluorescence-based capillary gel electrophoresis apparatus.
6. Selective co-immunopurification of the SSUs by the tagged eIFs and western blot analysis of the selective SSU enrichment
   ​NOTE: Use ~15 AU (260 nm) of the digested and sedimentation-segregated SSU fraction from step 1.4.11 to perform affinity purification using magnetic IgG beads. Save ~5% of the SSU fraction as input control (Input fraction, I). eIF4A-tagged (TIF1-TAP; Tandem Affinity Purification tag) yeast strain was used which also makes it possible to detect eIF4A by probing for the TAP-tag using anti-TAP antibody.
   1. Transfer 100 µL of magnetic IgG beads suspension (1 mg of the beads were used for each 15 AU (260 nm) of the lysate or fraction) into a new low protein binding 1.5 mL tube; collect the beads using magnetic rack and aspirate them.
   2. Wash the magnetic beads twice with 1 mL of buffer 1 by using sequential resuspension by pipetting and collection using the magnetic rack.
   3. After washing, collect and decant the beads, while keeping them on the magnetic rack.
   4. Add the SSU fraction to the washed beads and incubate the mixture for 4 h with rotation at 4 °C in a cyclomixer set at ~20 rpm.
   5. Collect the beads using the magnetic rack at 4 °C and save the supernatant (Flow-through fraction, FT).
   6. Wash the beads twice at 4 °C with buffer 1 supplemented with 4 mM DTT, each time rotating for 10 min in the cyclomixer and collecting and decanting the beads on the magnetic rack. Save the washes (W1 and W2 fractions).
   7. For an analytical application such as western blotting, elute the bound material under denaturing and reducing conditions by adding LDS (lithium dodecyl sulfate) polyacrylamide gel electrophoresis (PAGE) sample buffer with pH 8.5 to 1x and DTT to 2 mM.
   8. Heat the mixture at 95 °C for 5 min in a thermal block to finalize the elution.
   9. Collect the beads using the magnetic rack and recover the denatured eluate (E fraction) in a fresh low protein binding 1.5 mL microcentrifuge tube.
   10. Use the E fraction from the previous step to run a denaturing sodium dodecyl sulfate (SDS) PAGE immediately, or store the E fraction at -20 °C.
       NOTE: For a preparative collection of the TAP-tag-enriched translational complexes for any subsequent application, use an alternative elution approach employing Tobacco Etch Virus (TEV) protease. Refer to the Supplementary Table 1 for further details.
   11. To concentrate the dilute FT, W1 and W2 fractions, precipitate their material by adding 3x volumes of ice-cold acetone. Incubate the sample-acetone mix at -20 °C for 3 h.
   12. Pellet the precipitate by centrifuging the tubes at 13,000 x g for 10 min at 4 °C.
   13. Discard the supernatant and air dry the pellet in the open tubes at room temperature for 30 min.
   14. Dissolve the pellet in 7 µL of 1x LDS loading buffer supplemented with 2 mM DTT. Heat the samples in a thermal block set to 95 °C for 5 min.
   15. Load all I, FT, W1, W2, and E samples onto a 4%-12% w/v of acrylamide gradient, Bis-Tris polyacrylamide denaturing gel. Run the gel using 1x MES SDS (2- [N-mopholino]ethanesulfonic acid, sodium dodecyl sulfate) running buffer at 80 V, until the protein marker (10-250 kDa) resolves well and the lead dye reaches the bottom of the gel.
       NOTE: It is recommended to load serial dilutions of the WCL (whole cell lysate) (2-10 µg) on the gel as a control. It may take several attempts to achieve comparable loading of the gel across the fraction material.
   16. Transfer the protein content of the gel onto a polyvinylidene difluoride (PVDF) membrane by wet transfer method at 100 V for 1 h in a cold room as recommended by the western-blotting equipment manufacturer.
   17. Block the membrane using an appropriate blocking buffer (Phosphate Buffered Saline based) at room temperature for 1 h under constant shaking.
   18. Following manufacturer's instructions for antibody dilution, probe the membrane with anti-TAP-antibody for detecting the tagged eIF4A protein, anti-Pab1p antibody or anti-β-actin antibody (or any other desirable target) by overnight incubation of the membrane with Blocking Buffer (PBS)-diluted antibody (1:1,000 dilution) in a cyclomixer in a cold room.
       NOTE: 1:1,000 antibody dilution is a good starting point.
   19. Wash the membrane three times with 1x Phosphate Buffered Saline, 0.2% v/v Tween 20 (PBST) for 10 min each.
   20. Probe the membrane with fluorescently labeled secondary antibodies following manufacturer's instructions by incubating in a cyclomixer at room temperature for 1 h.
       NOTE: 1:20,000 antibody dilution is a good starting point.
   21. Wash the membrane three times with 1x PBST for 10 min each. Briefly rinse the membrane with deionized water, and then with absolute methanol. Dry and visualize the membrane in a fluorescent imaging system according to the manufacturer's instructions.
       ​NOTE: Staining for other proteins can be achieved by using secondary antibodies with dyes matching different fluorescent channels (such as in the eIF4A-TAP vs. β-actin pair used here), by sequential staining or stripping and staining the same membrane or cutting the membrane from a gel loaded with repeating pattern of fractions and separately probing each piece with respective antibodies (as in the Pab1p example used here).

2. Mammalian cell protocol

1. Mammalian cell culture and fixation
   1. In 2 T-175 flasks, grow HEK293 cells to 60%-70% confluence in Dulbecco's Modified Eagle Medium and 10% v/v Fetal Bovine Serum at 37 °C and 5% v/v carbon dioxide.
      NOTE: The complete media is made by adding 55 mL of commercial FBS into a 500 mL of commercially purchased DMEM with high glucose, containing L-glutamine, phenol red and sodium bicarbonate, but no HEPES or sodium pyruvate. Cell counts per T-175 flask at 70% confluence should be in the range of 1.7-2.0 x 10⁷.
   2. At least 3 h prior to the desired fixation time, replace the media of the T-175 flasks with precisely 30 mL of pre-warmed complete media and replace the flasks in a cell incubator.
      NOTE: Ensure that the fresh media is pipetted onto the opposite side of the flask to the cell monolayer to avoid cell detachment. Attempt to conduct the media exchange as quickly as possible, introducing minimal gas and temperature balance disturbance.
   3. Once the cell media has been replaced, prepare buffers and chemicals required for fixation. Prepare Dulbecco's Phosphate Buffered-Saline (DPBS) with 50 mM glycine by adding 10.2 mL of 2.5 M glycine stock to a 500 mL bottle of DPBS and mixing.
   4. Prepare a bottle of DMEM supplemented with 10% FBS as in step 2.1.1 to be used in non-sterile conditions and a 100 mL aliquot of 0.25% Trypsin-EDTA. Source an additional bottle of commercial DPBS pre-formulated with calcium chloride (CaCl₂) and magnesium chloride (MgCl₂).
      NOTE: The solutions can be stored at 4 °C for up to 2 weeks.
   5. Prepare an ice box to the brim with crushed water ice such that a T-175 flask can fit evenly on top and keep in the fume hood along with the prepared buffers, also on ice.
      NOTE: Due to the rapid responses of translation to any environmental change, all timings between the removal of the cell flasks from the incubator and addition of the formaldehyde solution must be minimized.
   6. To snap chill the cells, remove the T-175 flask from the incubator and firmly press it against the ice ensuring maximal surface contact. Inside the chemical fume hood, tilt the flask onto its side so that the media collects at the side opposite to the cells. Pipette 168 µL of 37% w/v formaldehyde directly into the pooled media (to a final concentration of 0.2% w/v). Immediately mix by gently rocking the flask back and forth, close and reposition the flask on ice, ensuring it is horizontal and the cells are covered evenly.
      CAUTION: Formaldehyde is a harmful substance with potential long-term adverse effects and also an irritant to both the respiratory system and skin. It should only be handled in a suitable chemical fume hood. Containers of formaldehyde must always be sealed when outside of the fume hood.
      NOTE: Ensure that the formaldehyde is added directly into the cell media and not to the flask wall. Step 2.1.6 should take less than 1 min.
   7. Incubate the flasks on ice for a further 10 min. Pour off the media into an appropriate waste container through the flask side opposite to the cells.
   8. Using a stripette, pipette in 30 mL of Dulbecco's Phosphate Buffered Saline without calcium and magnesium ions and additionally containing 50 mM glycine, gently on the side opposite to the cells. Mix by rocking the flask; return the flask to horizontal position and incubate for 10 min more on ice.
   9. Pour off the solution through the flask side opposite to the cells and gently add 7 mL of the standard 0.25% w/v Trypsin-EDTA solution to detach and resuspend the cells. Incubate the flask at room temperature for 5-10 min.
      NOTE: Ensure Trypsin-EDTA solution covers all the cells evenly. Use periodic gentle tilting and rocking to promote cell detachment.
   10. Relocate the flask vertically and using a stripette collect the detached cells by gently washing any remaining cells from the flask walls. Transfer the suspension into a 50 mL tube set on ice.
       NOTE: Fixed cells can become more fragile; do not pipette intensely or more than what is required to detach the cells from the flask wall.
   11. Immediately supplement the collected cell suspension with 20 mL of complete media (the non-sterile ice-cold media with 10% FBS) and mix by gently flipping the tube.
       NOTE: The complete cell culture media (including 10% FBS) is added to neutralize the trypsin, preventing further damage to the cell membranes and cell disintegration.
   12. Pellet the cells by centrifuging the tube at 100 x g for 5 min and 4 °C. Cell pellet must be clearly visible.
   13. Pour off the media and gently resuspend the cell pellet in 10 mL of ice-cold DPBS with Ca²⁺, Mg²⁺, and without glycine.
   14. Repeat step 2.1.12.
   15. Pour off the wash buffer and gently resuspend the cell pellet in 800 µL of ice-cold DPBS with Ca²⁺, Mg²⁺, without glycine, on ice. Transfer the resuspended cells into a new low protein binding 1.5 mL microcentrifuge tube.
   16. Centrifuge the tube at 100 x g for 3 min and 4 °C. Carefully discard the supernatant using a 1 mL pipettor. At this stage, the cell pellet can be frozen at -80 °C or proceed to the cell lysis step.
       NOTE: Frozen cell pellets can be stored at -80 °C up to 1 year. We found that cell pellet freezing facilitates subsequent lysis and recommend freezing even if longer term storage is not planned.
2. Mammalian cell disruption and cytosol collection
   1. In a biosafety cabinet, add 300 µL of the lysis buffer based on nonionic, nondenaturing detergent and 7 µL of 40 U/µL RNase inhibitor. Mix well by pipetting using a 1 mL tip.
   2. Carefully attach a 25 G needle to a 1-3 mL syringe and vigorously pipette the mixture, using at least seven slow upward intake and fast downward exhaust strokes.
   3. Discard the syringe and needle into a sharps bin and repeat the procedure using a 0.3 mL syringe equipped with a 31 G needle.
   4. Discard the syringe and needle into a sharps bin. Centrifuge the tubes at 4 °C, 12,000 x g for 5 min to pellet the cell debris.
   5. Transfer the supernatant into a new low protein binding 1.5 mL microcentrifuge tube. Store both, the cell debris (for control purposes) and the resultant clarified cell lysate at -80 °C.
      ​NOTE: Optical density of the lysate ranges between 25-30 AU₂₆₀ when two T-175 flasks are combined and the recommended volumes followed. The lysates and cell debris can be stored at -80 °C up to 1 year.
3. Separation of the fixed (poly)ribosomal complexes from the non-translated fractions of the cytosol
   1. Prepare linear 15%-45% w/v sucrose gradients in 13 mL thin wall polypropylene tubes, 14 x 89 mm, using freeze-thaw method generally as described in step 1.3.1 of the yeast protocol, but using buffer 2 (Figure 2a).
      NOTE: Thaw the gradients overnight in a cold room at 4 °C the night prior to the fractionation.
   2. Load 150-250 (maximally 300) µL of the cell lysate from the previous step 2.2.5 onto the balanced gradients. Store the remaining lysate at -80 °C and use for control purposes.
      ​NOTE: Here an example of sedimentation-based segregation into polysomal, ribosomal and 'free' SSU fractions is provided. Refer to the provided Supplementary Table 1 for an alternative approach.
   3. Ultracentrifuge the tubes in a medium volume swing-bucket rotor at 4 °C, average g-force 178,305 x g (k-factor 143.9) for 1 h 45 min.
   4. 30 min prior to the spin completion, set up and baseline the gradient fractionator, as described in the yeast protocol steps 1.4.7-1.4.9.
   5. Fractionate the gradients generally as described in the yeast protocol steps 1.4.10-1.4.11.
      ​NOTE: This step will separate polysomal, ribosomal and 'free' SSU fractions. Polysomal fractions may be used in polysome profiling experiments.
   6. Immediately transfer the collected fractions on ice and if not further processed, store at -80°C up to 6 months.
      NOTE: If the fraction collector tube change is synchronized with the on-line fraction identification and segregation, we recommend using up to 800 µL fractions (collection time of 32 s per fraction at 1.5 mL/min). If the fractionation is performed without using the in-line absorbance readout, it is recommended using 250-500 µL fractions (10-20 s per fraction at 1.5 mL/min). Following separation, the fractions can be used for immunopurification, electron microscopy, denaturing PAGE and western blotting straight away, or subjected to crosslink reversal for subsequent RNA and/or proteomics analyses.

结果

翻译复合物对缓冲液的离子组成较为敏感，这一点在超速离心过程中尤为重要，因为该过程需评估沉降特性。因此，我们使用从研磨后的未经固定的酵母材料中提取的澄清裂解液，测试了多种沉降缓冲液，以筛选出最适合分离翻译复合物并分辨核糖体亚基（SSU，LSU）、单体核糖体（RS）及多聚核糖体的梯度条件。所有缓冲液均基于核心组分：25 mM HEPES-KOH（pH 7.6）和 2 mM DTT。在不同缓冲液中进一步调整了 KCl、MgCl₂、CaCl₂ 和 EDTA 的浓度（图 2a），这些组分根据需要分别在上样前加入裂解液，或在制备蔗糖梯度前加入梯度缓冲液中。

在缓冲液1和2中均获得了分辨良好的翻译复合物。缓冲液1在小核糖体亚基（SSUs）的分离效果上略优（图2a）。省略MgCl₂并添加EDTA（缓冲液3、4）导致大多数多聚核糖体的高沉降特性丧失，可能发生了部分解聚（图2a）。尽管添加2.5 mM CaCl₂使多聚核糖体峰略微更均一，但改善程度有限，且与缓冲液1和2相比，此时多聚核糖体物质的总量有所减少（图2a）。因此，我们选择缓冲液1作为首选的工作缓冲液。

图2：翻译复合物提取的缓冲条件及固定对稳定性的评估。图中显示了通过10%–40% w/v蔗糖梯度分离的总酵母细胞裂解液在260 nm处测得的紫外吸光度谱图。(a) 单价和二价盐以及镁离子螯合对未固定酵母细胞提取物沉降行为的影响。红色和灰色线条代表典型的重复实验结果。(b,c) 来源于未固定（灰色线条）、2.2%（黑色线条）和4.4% w/v甲醛固定的酵母细胞（黑色虚线）裂解液的比较。(d) 与未固定HEK 293T细胞（灰色线条）相比，经优化的0.2% w/v甲醛固定（黑色点划线）对多聚核糖体的稳定作用。请点击此处查看该图的放大版本。

接下来，我们检测了使用不同浓度甲醛固定对多聚核糖体稳定性的影响。在细胞材料、缓冲液、细胞处理及时间控制等其他条件完全相同的情况下，我们比较了未固定细胞与经2.2%和4%（w/v）甲醛固定的细胞所提取的材料（图2b，c）。结果表明，2.2%（w/v）甲醛更适用于固定，因为它在很好地维持多聚核糖体方面表现优异，这一点可通过多聚核糖体与单体核糖体的比值来判断（图2b），同时与4%（w/v）甲醛相比，其并未降低核糖体材料的总体得率；而后者则表现出明显的过度固定迹象（图2c）。

对于来源于哺乳动物细胞的材料，由于基于去污剂的提取需要较大的裂解缓冲液体积与细胞体积之比，因此使用了缓冲液2（图2a）。该条件在蔗糖梯度离心中可产生分辨良好的翻译复合物（图2d）。值得注意的是，此处使用的甲醛浓度较低，仅为0.2%（w/v），因为较高浓度会导致多核糖体及核糖体物质显著丢失（数据未显示）。与酵母细胞实验结果相似，交联稳定的样品显示出多核糖体更好的保存效果以及更高的多核糖体与单核糖体比率（图2d）。

接下来，我们检测了所选的甲醛固定条件是否足以通过交联作用有效稳定多聚核糖体组分中正在活跃翻译的mRNA，从而确认多聚核糖体产量的提高并非仅仅源于酶功能和翻译延伸进程的抑制。我们使用EDTA和高单价盐（KCl）来破坏多聚核糖体和核糖体的稳定性。这些试剂被添加至澄清的酵母细胞裂解液中，并分别在后续所有缓冲液及蔗糖梯度中，叠加于缓冲液1的组成之上。

事实上，15 mM EDTA 对来自固定细胞的多聚核糖体组分的去稳定作用较弱（图3a），证实交联复合物更为稳定。EDTA 的去稳定效应可通过提高甲醛浓度在一定程度上得以克服，因为经 4% w/v 甲醛固定的细胞样品能更好地抵抗结构解聚（图3a）。然而，当 EDTA 浓度升高至 50 mM 时，在固定与未固定条件下，大多数翻译复合物均发生去稳定化，这可从物质沉降速度变慢以及缺乏清晰峰形中推断得出（图3b）。这一现象可归因于结构的部分解折叠及整体致密性的降低，而非多聚核糖体组分从 mRNA 上的完全解离。即便在此情况下，交联样品仍表现出更快的沉降速率（图3b）。

图3：影响 体内 酵母甲醛固定对多聚核糖体稳定性的影响 缓冲液1（见正文和 图2a) 在所有实验中均被使用。数据类型和绘图方式如文中所述 图2 图注。 (a) 在非交联细胞（灰色线）、2.2%（黑线）和4%（黑虚线，w/v）甲醛交联细胞来源的多聚核糖体稳定性中，比较在细胞裂解液及后续缓冲液中添加15 mM EDTA的影响。b）与（a)，但需添加50 mM EDTA，并排除4% w/v的甲醛固定细胞。c）与（a），但需添加500 mM KCl，并排除2.2%（w/v）的甲醛固定细胞。 请点击此处以查看此图的放大版本。

与EDTA的作用类似，在500 mM KCl条件下，我们发现4% w/v的甲醛固定显著提高了稳定性（图3c）。此种情况下观察到的致密性明显降低，也可归因于核糖体复合物组分的部分脱离，而非其从RNA上的完全解离。总体而言，来自甲醛固定细胞的多聚核糖体表现出更强的抗解折叠和结构去稳定能力，这与这些复合物内部形成了额外的共价键一致。

在促进生长的条件下，mRNA 可以迅速启动翻译，导致多个核糖体聚集在同一 mRNA 分子上，形成称为多聚核糖体（polyribosomes）或简称多聚体（polysomes）的结构。多聚体可通过蔗糖梯度超速离心进行分离，其沉降行为取决于其级数（即 mRNA 上同时结合的核糖体数量）。当翻译受到抑制时，核糖体无法及时进入下一轮翻译，导致多聚体发生（部分）“解聚”，表现为多聚体向较低级数迁移的模式性偏移，并伴随单体核糖体（monosomes）的积累^4,26。

葡萄糖饥饿可提供一种能够在多核糖体分布水平上可视化的翻译响应模型。葡萄糖耗竭会在酵母中引发最显著且迅速的翻译抑制效应之一^1,3,40。先前的研究表明，在葡萄糖耗竭后1分钟内，即可发生多核糖体的减少、单核糖体的积累以及翻译起始的抑制⁴。在重新补充葡萄糖5分钟内，翻译迅速恢复，并伴随多核糖体的明显增加^3,4。此外还观察到，当细胞暴露于葡萄糖浓度为0.5%（w/v）或更低的培养基中时，翻译受到抑制；而在葡萄糖浓度为0.6%（w/v）或更高时，则未观察到抑制效应。

因此，我们希望确定所采用的固定条件是否适用于保存葡萄糖应激反应动态过程中存在的翻译水平差异，这种差异可通过多聚核糖体与单核糖体的比值进行评估。我们将生长在高葡萄糖（添加2.00% w/v）培养基中、处于对数中期的细胞材料，与转移至无添加或低添加葡萄糖（分别为0.00% 或 0.25% w/v）培养基中10分钟后的细胞材料进行了比较。固定处理采用2.2% w/v甲醛进行，同时在对照组（非饥饿组；快速更换为含2% w/v添加葡萄糖的标准培养基，孵育10分钟后固定）和饥饿10分钟组（快速更换为相同培养基但含低浓度0.25% w/v或无添加葡萄糖，孵育10分钟后固定）细胞中平行开展。

与先前的研究结果一致，我们观察到酵母细胞在葡萄糖饥饿应激条件下会强烈抑制翻译（图4a）。无论未添加葡萄糖还是低葡萄糖条件均诱导了多聚核糖体的解聚，其中低葡萄糖添加条件下保留的多聚核糖体略多，但差异明显。因此，酵母对葡萄糖去除的响应可能并非完全“开启”或“关闭”式的反应，而是逐步调节的。正如预期，甲醛交联具有稳定作用，固定后的细胞中多聚核糖体物质在饥饿与非饥饿细胞之间表现出更明显的区分度，可能更好地保留了响应过程中的动态范围（图4b）。有趣的是，在固定细胞的样本中，低葡萄糖添加浓度所导致的多聚核糖体丰度与未添加葡萄糖条件之间的差异显著优于未固定的细胞（图4a）。这强烈表明甲醛固定法适用于保存和捕捉高度动态过程（如翻译响应期间）中相对微小且短暂的平衡变化。

图 4：在葡萄糖饥饿条件下捕获酵母翻译过程的快速变化。 所有实验均使用缓冲液 1（见正文及图 2a）。数据类型与绘图方式参见图 2图注。(a) 来自非饥饿（灰色线）、限制性葡萄糖饥饿（添加 0.25% w/v 葡萄糖，处理 10 分钟；棕色线）和葡萄糖耗尽（不添加葡萄糖，处理 10 分钟；红色线）的未固定酵母细胞裂解液。(b) 同 (a)，但使用 2.2% w/v 甲醛固定的细胞。请点击此处查看该图的高清版本。

利用蔗糖梯度沉降法（“多聚核糖体分析”）监测与正在活跃翻译的mRNA结合的核糖体状态，是一种广泛应用的技术^26,27,28。结合定量微阵列分析，以及近年来的高通量测序技术^28,44，多聚核糖体分析可在全转录组范围内提供有关核糖体结合mRNA的信息。在蛋白质生物合成研究领域，传统上普遍认为：mRNA存在于多聚核糖体中，表明其正积极参与翻译过程。进一步的推论通常（但并非总是）成立：对于特定长度的mRNA，其上结合的核糖体越多（即多聚核糖体的阶数越高），该mRNA参与翻译的活跃程度就越高。因此，将多聚核糖体组分与其他物质分离，有助于富集正在活跃翻译的RNA。在核糖体印迹分析方法中，尤其是TCP-seq^10,38,39，该方法可生成来源于扫描复合物、起始密码子复合物和终止密码子复合物的游离小亚基（SSU）的独立群体，此时额外去除那些不能与完整单体核糖体或多聚核糖体共沉降的核糖体亚基，可能提供更深入的信息。

因此，我们将“非翻译状态”的mRNPs（例如游离的SSU，即与单个SSU结合的mRNA，或未结合mRNA的SSU）与“正在活跃翻译”的mRNA群体分离开来。为实现这一目的，我们假设与单个（单体）或多个核糖体（多聚核糖体）结合的mRNA可能正处于活跃翻译状态。这类复合物因其具有较高的沉降系数，可与其他成分分离。我们还建议将“正在活跃翻译”的mRNA群体分离至蔗糖垫层（50% w/v 蔗糖），而非直接在离心管管壁上沉淀收集。将快速沉降的复合物离心进入垫层，可使我们通过吸光度谱图监测分离过程，并获得比沉淀后重新溶解更高的可溶性、非聚集且未变性材料得率^10,38。

总体而言，为了纯化单个小亚基（SSUs）、核糖体、双体（disomes）以及可能存在的高级有序紧密排列的多聚核糖体，将固定的澄清裂解液进行了两步超速离心处理（图5）。在第一次蔗糖梯度离心中，超速离心导致游离的小亚基（SSUs）和大亚基（LSUs）分离并位于梯度上部（蔗糖质量体积比为10%–20%）的区域，而交联的翻译产物池（包括多聚核糖体以及与一个完整核糖体结合的mRNA）则富集于梯度底部（蔗糖质量体积比为50%）的区域。图5a). 随后浓缩含有翻译后mRNA池的蔗糖层下部50% w/v部分，并用RNase I消化其RNA，再进行第二次蔗糖梯度超速离心，以分别获得小亚基（SSU）、大亚基（LSU）、核糖体复合物（RS）、RNase抗性的二聚体核糖体（DS）以及少量的高阶核酸酶抗性多聚核糖体 (图5b). 通过醋酸铀负染色并使用透射电子显微镜成像，确认了各沉降阶段所分离复合物的身份图5).

图 5：从非翻译RNA中分离出全部已翻译的RNA组分。（a,c）示意图（左）及相应的代表性结果（右；数据类型和绘图方式见图 2图注说明），其中（a）为首次不连续蔗糖梯度分离，将非翻译的胞质组分（包括游离的小亚基SSU）与通过与核糖体及多聚核糖体共沉降而鉴定出的已翻译mRNA组分进行分离；（c）为经RNase I可控消化并穿过第二次线性蔗糖梯度超速离心后，将从已翻译mRNA组分中释放出的各个核糖体复合物进一步分离为小亚基（SSU）、大亚基（LSU）、核糖体（RS）以及核酸酶抗性双体（DS）组分。实验中分别使用了高（15 AU₂₆₀）和低（8 AU₂₆₀）上样量的非饥饿处理消化样品，以展示在关注微量组分时增加超速离心上样量的可能性。此外也可识别出更高阶的核酸酶抗性多聚核糖体（例如，本示例中可见三聚体）。（b,d）分别为来自（a,c）的醋酸铀染色组分的代表性透射电镜（TEM）图像。请点击此处查看该图的放大版本。

为了检验固定方案对瞬时核糖体相关蛋白（特别是起始因子eIFs）保留的适用性，我们检测了不稳定且动态结合于核糖体的起始因子eIF4A是否在各核糖体组分中发生共沉淀。我们利用带有TAP标签的eIF4A酵母菌株（TIF1-TAP），检测了固定后样品中eIF4A在核糖体组分中的存在情况。 与 使用抗TAP抗体对非固定细胞进行检测，并以Pab1p作为额外的RNA结合对照，通过SDS-PAGE电泳及随后的蛋白质印迹（western blotting）比较其丰度（图6).

图6：翻译复合物中瞬时蛋白的稳定化 体内 甲醛固定 (a,b) (上方图示) 全细胞裂解液（WCL）的（a) 未固定的和（b) 经2.2%甲醛固定的eIF4A-TAP酵母细胞，通过超速离心分离，并按所述方法进行观察 图2 图注。底部图示) 对相应蔗糖梯度分离后各组分进行Western印迹成像（上图），并以全细胞裂解液（WCL）作为对照。ceIF4A 或 Pab1p 在固定与非固定样品各组分中的平均丰度比值。根据 (a,b) 的数据（底部图），计算了 eIF4A（黑色柱）和 Pab1p（灰色柱）在第 2、3 组（SSU，LSU）、第 4、5 组（RS，轻度多聚核糖体）以及第 6、7 组（重度多聚核糖体）中的相对比例（归一化至第 2–7 组信号总和），并求得其固定样品与非固定样品的比值。误差线表示将合并的组分（虚线框）视为重复样本时，比值相对于平均值的标准差。 请点击此处以查看此图的放大版本。

与这两种蛋白在细胞中含量较高相一致，我们在未固定细胞的全细胞裂解液（WCL）以及沉降较慢的组分中均观察到较强的信号（图6a，底图）。我们还在固定细胞的全细胞裂解液中检测到大量这些蛋白，表明交联材料的提取效率较高，且未出现意外损失（图6b，底图）。然而，与未固定细胞不同的是，来自固定细胞的材料显示，在沉降较快的核糖体组分中，eIF4A 相对于 Pab1p 的相对丰度升高（图6c）。该结果提示，在甲醛交联的样品中，eIF4A 与多聚核糖体的结合更为牢固。

在确认交联对eIF4A在核糖体组分中存在具有阳性且特异的稳定作用后，我们利用带有eIF4A标签（TIF1-TAP）的酵母菌株的固定材料，通过磁性IgG珠进行亲和纯化，以捕获并富集含eIF4A的复合物。我们在蔗糖梯度第一次沉降后，对WCL、游离SSU和多聚核糖体（翻译中的mRNA池）组分进行了亲和富集（例如酵母实验方案的第1.3节），并在RNase I处理将翻译池解离为各个复合物后，对第二次沉降所得的SSU、LSU和RS组分也进行了富集（例如酵母实验方案的第1.4节）（图7）。除LSU组分外，在所有情况下，与起始材料中的β-actin（上样量，I）相比，我们均能在纯化后的组分（洗脱液，E）中观察到eIF4A的选择性富集（图7）。

图7选择性免疫纯化 体内 由瞬时结合的eIF4A稳定化的甲醛交联翻译复合物 示意图展示了不同翻译复合物及eIF4A表位的来源，包括未分级的eIF4A-TAP酵母细胞澄清WCL；通过第一次超速离心分离出的游离SSU和翻译RNA池（多聚核糖体）；经RNase I消化释放出SSU、LSU和RS组分，并通过第二次超速离心进行分离（见正文）。Western印迹图像显示了各组分中eIF4A的丰度，与同时染色的β-actin对照的丰度进行比较。 请点击此处以查看此图的放大版本。

补充表 1。 请点击此处下载该表格。

讨论

甲醛固定是一种方便且常用的方法，可实现生物分子的快速体内交联^{10,36,45,46,47,48}。与其他可能的生物分子靶标相比，成功捕获翻译复合物需要在细胞或其他材料快速冷冻的同时立即进行固定。若不及时稳定，多种与翻译相关的过程可能继续进行，导致复合物分布偏离未受干扰的体内状态⁴⁹。与其他翻译抑制和核糖体复合物稳定方法相比，甲醛能够迅速穿透细胞膜，并以非特异性方式形成交联，因而有望最大限度地保留翻译复合物中间体的多样性，使其更接近天然分布状态⁵⁰。

本文所述方法已在酵母和哺乳动物细胞中建立并优化，其他研究团队也已在此基础上开发出适用于更多样化生物材料的方法，例如在完整脊椎动物（如斑马鱼胚胎）中的应用^{10,38,39,49,51,52}。尽管这些研究共同证实了该方法的通用性和广泛适用性，但由于需要进行优化和调整，翻译复合物的快速甲醛交联在转移至新型生物材料时仍被认为具有一定难度。

该方法成功的关键要求之一是对甲醛浓度以及细胞收集和破碎技术进行重新优化。通透性较差、体积小且呈圆形的酵母细胞需要更高浓度的甲醛（至少高10倍）并对固定后的细胞进行物理破碎。相比之下，培养中的大型扁平贴壁哺乳动物细胞容易发生过度固定，因此在固定过程中需轻柔操作，而固定复合物的提取则可通过使用去垢剂进行化学裂解膜结构来完成。交联不足可能导致较不稳定或寿命较短的中间产物解离或渗漏至后续状态；而交联过度则可能对核糖体组分的分离与研究产生不利影响，并引发选择性偏差，例如导致较重复合物更显著的损耗。据我们观察，即使是微小的变化，例如所用贴壁人源细胞的类型不同，也可能影响交联复合物的回收产量，因而需要重新优化交联方案。我们还可以预见，具有显著不同通透性特征的细胞（如植物细胞）将需要对固定条件进行更广泛的额外优化⁵²。然而，很难设想有任何类型的生物材料会完全不适用于该方法。

与哺乳动物细胞固定方案相关的一个考虑因素是用作输入的细胞材料的密度和数量。建议细胞持续生长至少2天，期间不进行传代培养或其他干扰，以避免对外部细胞翻译动态产生影响。该建议适用于大多数细胞类型，但对于大多数贴壁细胞，保持汇合度不超过70%可确保避免显著的接触抑制效应，此类效应可能对翻译速率产生负面且不可预测的影响。

甲醛固定的另一个有趣且可能具有独特便利性的特征源于其无选择性的反应性，即对混合分类系统中翻译复合物的稳定作用。细菌，尤其是线粒体、叶绿体以及各种细胞内寄生生物的翻译复合物，历来难以通过特异性的翻译抑制剂进行靶向研究。相比之下，在TCP-seq数据中，映射到线粒体转录组的足迹序列可被清晰观测到^38,39,50。一个值得关注的后续发展方向是利用该方法研究整个微生物群落（如土壤、水体或肠道样本）中的翻译过程，因为在这些复杂环境中，若采用其他方法实现可靠且快速的翻译终止及复合物稳定，将面临较大困难。

还应指出，对于最复杂的材料（如坚硬和/或体积较大的组织），在细胞破裂及材料均质化后可立即使用甲醛进行固定，且并无任何限制。该方法已被广泛用于在利用特定小分子抑制剂稳定翻译复合物时，消除细胞通透性带来的延迟。^33,53,54,55鉴于甲醛固定在传统上一直被使用，并取得了极好的效果 离体/体外 样品稳定化在电子显微镜等应用中的作用^45,56,57,58在这种情况下，我们可以预期负面效应会更少，特别是与从充分固定的细胞中提取翻译复合物效果不佳相关的问题。

我们的研究结果证实，快速甲醛固定法可用于稳定高度瞬时的复合物，例如包含 eIF4A 的复合物。值得注意的是，与哺乳动物不同，酵母中的 eIF4A 与帽结合复合物 eIF4F 的结合要弱得多，因此总体上与翻译复合物的结合也较弱。在酵母中，任何对核糖体材料进行的较彻底纯化过程通常都会导致 eIF4A 的丢失^{29,59,60,61,62,63}。然而，在经过 in vivo 固定的酵母样品中，可以在所有预期存在 eIF4A 的翻译复合物组分中实现其可靠富集。先前发表的 Sel-TCP-seq 数据已展示了与核糖体结合更强的 eIF2 和 eIF3 的富集情况（同时也揭示了瞬时发生的共翻译蛋白复合物组装）³⁹。因此，该方法适用于检测与翻译复合物结合较强和较弱的各类组分。

综上所述，我们介绍了一种可用于深入研究翻译起始阶段变化的方法，尤其适用于需要最小程度干扰mRNA上核糖体分布的情况。重要的是，该方法适用于稳定翻译复合物中相对不稳定且动态的组分（如eIF4A），并在经过必要优化后可广泛使用。我们还提供了证据，表明甲醛固定在研究翻译过程快速动态变化的情境中具有实用价值，从而为研究细胞对环境变化或应激条件的快速反应等课题开辟了新的方向。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
酵母提取物	Merck, Sigma-Aldrich	70161
蛋白胨	Merck, Sigma-Aldrich	70178
D-葡萄糖（右旋糖）	Merck, Sigma-Aldrich	49139
硫酸腺嘌呤	Amresco	0607-50G
甲醛溶液	Merck Sigma-Aldrich	F11635-500ML	ACS级试剂，37 wt. % 溶于 H2O，含10-15%甲醇作为稳定剂（防止聚合）
RNaseOUT™ 重组核糖核酸酶抑制剂	Invitrogen™ byThermo Fischer Scientific	10777019
cOmplete™，无EDTA蛋白酶抑制剂混合物	COEDTAF-RO Roche by Merck	11873580001
氯化镁溶液	(Merck/Sigma-Aldrich)	M1028
乙二胺四乙酸溶液	(Merck/Sigma-Aldrich)	E7889
Ambion™ RNase I，克隆型，100 U/µL	Ambion	AM2294
SUPERase•In™ RNase抑制剂（20 U/μL）	Invitrogen™ by Thermo Fisher Scientific	AM2694
酸性苯酚:氯仿:异戊醇 125:24:1（pH 4.0-5.0）	(Merck/Sigma-Aldrich)	P1944-100ML
Dynabeads™ 羊抗小鼠IgG磁珠	Invitrogen™ by Thermo Fisher Scientific)	11033
醋酸钠（3 M），pH 5.5	Invitrogen™ by Thermo Fisher Scientific)	AM9740
糖原（5 mg/ml）	Invitrogen™ by Thermo Fisher Scientific)	AM9510
乙醇，纯级	Merck; Sigma Aldrich	E7023
Amersham™ Hybond® P 蛋白印迹膜，PVDF	Merck	GE10600023	PVDF膜用于蛋白印迹
Bolt™ 4 至 12%，Bis-Tris，1.0 mm，Mini Protein Gel	Invitrogen™ by ThermoFischer Sientific	NW04120BOX	蛋白凝胶
4X Bolt™ LDS 上样缓冲液	Invitrogen™ by ThermoFischer Sientific	B0007	LDS上样缓冲液
Precision Plus Protein™ Kaleidoscope™ 预染蛋白标准品	BioRad	1610375	蛋白分子量标准
20X Bolt™ MES SDS 电泳缓冲液	ThermoFischer Scientific	B0002	PAGE电泳缓冲液
Intercept® (PBS) 封闭缓冲液	LI-COR	927-70001	Odyssey封闭缓冲液（PBS）
IRDye® 800CW 羊抗小鼠IgG二抗	LI-COR	92632210
IRDye® 800CW 羊抗兔IgG二抗	LI-COR	92632211
TAP标签多克隆抗体	Invitrogen™ by ThermoFischer Sientific	CAB1001
抗β-肌动蛋白抗体	Abcam	ab8227
蔗糖	(Merck/Sigma-Aldrich)	84097	BioUltra，分子生物学级，≥99.5%（HPLC）
DL-二硫苏糖醇溶液	(Merck/Sigma-Aldrich)	43816	BioUltra，分子生物学级，~1 M 溶于 H2O
Terumo 1CC/mL 注射器	Terumo Syringe	878499
氯化钾	(Merck/Sigma-Aldrich)	60128
HEPES	(Merck/Sigma-Aldrich)	H3375
DMEM高糖培养基（Dulbecco改良Eagle培养基）	Sigma Aldrich	D5796
胎牛血清	Sigma Aldrich	12003C
胰蛋白酶-EDTA（0.05%），含酚红	Gibco	25300062
含钙镁的磷酸盐缓冲液（Dulbecco's Phosphate Buffered Saline）	Sigma-Aldrich	D8662
甘氨酸	Sigma-Aldrich	G7126
Tris盐酸盐	Merck/Sigma-Aldrich	10812846001
十二烷基硫酸钠	Merck/Sigma-Aldrich	436143
IGEPAL CA-630	Merck/Sigma-Aldrich	I3021
Rnasin 核糖核酸酶抑制剂	Promega	N2111
不锈钢研磨罐	Retsch	02.462.0059
MM400 混合研磨仪	Retsch	20.745.0001
梯度分馏仪	Brandel	BRN-BR-188
Thermomixer R	Eppendorf	Z605271
Nanodrop 分光光度计	Thermo Fisher Scientific	ND-2000
0.5 ml 带锁装置的微量离心管	Eppendorf Safe-Lock	30121023
Mini Gel 电泳槽	(Thermo Fisher Scientific)	A25977	PAGE电泳槽
5 mL 开口薄壁超透明离心管，13 x 51 mm	Beckman-Coulter	344057
13.2 mL 认证无菌开口薄壁聚丙烯离心管，14 x 89 mm - 50支装	Beckman-Coulter	331372
Amicon Ultra-0.5 超滤装置	Merck	UFC5030	Ultracel-30 再生纤维素膜，0.5 mL 样品体积
Thermo Sorvall Evolution RC 地面超速离心机	Cambridge Scientific	15566
Beckman Coulter Optima L-90K	GMI	8043-30-1191
Nunc EasYFlask 175 cm² 培养瓶	Thermofisher Scientific	159910
Falcon 50 mL 锥形离心管	Thermofisher Scientific	14-432-22
25 mL 移液管	Sigma-Aldrich	SIAL1250
10 mL 移液管	Sigma-Aldrich	SIAL1100
DNA低吸附管	Eppendorf	30108051
5810 R 冷冻离心机	Eppendorf	EP022628188	适用于50 mL离心管
轨道摇床培养箱	Ratek	OM11
Frezco 17 微型离心机	Thermofisher Scientific	75002402
Eppendorf DNA低吸附管	Merck/Sigma-Aldrich	EP0030108051
Eppendorf® Protein LoBind 管	Merck/Sigma-Aldrich	EP0030108116
SW 41 Ti 摆动桶转子	Beckman-Coulter	331362
Heracell™ 150i CO2 培养箱，150 L	Thermofisher Scientific	51026282
0.3 mL 超细II型短胰岛素注射器	BD Medical	328822
3 mL 带鲁尔锁扣头的注射器	BD Medical	302113
25 G x 16 mm 皮下注射针头	Terumo	TUAN2516R1