在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析
Summary
polysome 分析技术的总体目标是分析个体基因或转录基因在蛋白质合成过程中的平移活动。该方法对健康和多人疾病的蛋白质合成调控、翻译活化和抑制的研究具有重要意义。
Abstract
正确的蛋白质表达在适当的时间和正确的数量是正常细胞功能和生存在一个快速变化的环境的基础。长期以来, 基因表达研究以转录水平的研究为主。然而, 基因的稳态水平与蛋白质的生产没有很好的关联, 基因的可译性因条件而变化很大。在某些有机体, 象寄生虫利什曼原虫, 蛋白质表达在翻译水平上主要被调控。最近的研究表明, 蛋白质翻译失调与癌症、新陈代谢、神经变性和其他人类疾病有关。Polysome 分析是研究蛋白质翻译规律的有力方法。它允许测量个体基因的平移状态或在全基因组范围内检查翻译。这种技术的基础是分离 polysomes, 核糖体, 他们的亚基和自由基因在离心的细胞质裂解过程中通过蔗糖梯度。在这里, 我们提出了一个通用的 polysome 分析协议, 用于三种不同的模型-寄生虫利什曼原虫少校, 培养的人类细胞和动物组织。利什曼原虫细胞在悬浮体中自由生长, 培养的人细胞生长在粘附单层, 而小鼠睾丸代表动物组织样本。因此, 该技术适用于所有这些来源。polysomal 分数分析的协议包括通过 rt-pcr 检测个体 mRNA 水平、qPCR 蛋白和用电泳分析核糖体 rna。通过对基因与核糖体在转录水平上的研究, 通过对核糖体相关蛋白的分型质谱分析, 可以进一步扩展该方法。该方法可以很容易地调整到其他生物模型。
Introduction
细胞基因表达调控由转录、转录后水平和翻译后机制控制。在深 RNA 测序方面的进展允许在一个空前的水平上研究基因组范围内的稳态 mRNA 水平。然而, 最近的研究发现, 稳态 mRNA 水平并不总是与蛋白质生产相关1,2。一个单独的成绩单的命运是非常复杂的, 取决于许多因素, 如内部/外部刺激, 压力,等。在蛋白质合成过程中, 基因表达调控为在变化条件下快速反应提供了另一层表达控制。Polysome (或 “polyribosome”) 剖面分析, 积极翻译核糖体的分离和可视化, 是研究蛋白质合成调控的有力方法。虽然, 它的第一个实验应用出现在二十世纪六十年代3中, polysome 分析目前是蛋白质翻译研究4中最重要的技术之一。单基因可由多个核糖体转化导致形成 polysome。转录可以停滞在核糖体与放线菌酮5和基因包含不同数量的 polysomes 可以在 polysome 分馏过程中分离的蔗糖梯度离心 6, 7 ,8,9. polysomal 分数的 RNA 分析然后允许测量基因组范围内单个基因的平移状态的变化, 在不同的生理条件下4,7,10. 该方法还用于揭示 5 ‘ UTR 和 3 ‘ UTR 序列在 mRNA 可译性的控制中的作用11, 检查 miRNAs 在平移抑制中的角色12, 揭开核糖体合成13 的缺陷, 并了解核糖体相关蛋白与人类疾病的作用14,15。在过去的十年中, 在翻译过程中对基因表达调控的作用越来越大, 这说明了它在人类疾病中的重要性。在癌症、代谢和神经退行性疾病中进行平移控制的证据非常惊人15,16,17,18。例如, eIF4E-dependent 平移控制的失调有助于自闭症相关的赤字15和 FMRP 参与停滞的核糖体基因链接到自闭症14。因此, polysomal 分析是研究多种人类疾病的翻译调节缺陷的重要工具。
不同生理条件下 polysomal 分数的蛋白质分析, 对翻译过程中核糖体相关因素的作用进行了剖析。polysome 分析技术已用于许多物种, 包括酵母, 哺乳动物细胞, 植物和原生动物10,19,20,21。像锥和利什曼原虫这样的原生动物寄生虫表现出有限的基因表达转录控制。他们的基因组被组织成 polycistronic 基因簇, 缺乏启动子调控的转录22。相反, 发展基因表达主要控制在蛋白质的翻译水平和 mRNA 稳定性的 trypanosomatid 物种23,24。因此, 在缺乏转录调控的情况下理解平移控制对这些生物体尤为重要。Polysomal 分析是研究利什曼原虫25、26、27、28中基因表达转录后水平调控的有力工具。
通过实时定量 PCR (RT-qPCR) 和转录的下一代测序以及蛋白质组学技术检测个体基因水平的最新进展, 将 polysomal 分析的解决方案和优势带到了一个新的层次。通过对单个 polysomal 分数的分析, 结合蛋白质组学分析的方法, 可以进一步扩大对各细胞的使用, 以监测整个基因型范围内体细胞的平移状态。这使得在不同的生理和病理条件下, 识别新的分子玩家调节翻译。在这里, 我们提出了一个通用的 polysome 分析协议, 用于三种不同的模型: 寄生虫利什曼原虫少校, 培养的人类细胞和动物组织。本文就不同生物体的细胞裂解物的制备、梯度条件的优化、RNase 抑制剂的选择、qPCR、印迹和 RNA 电泳等方面的应用提出建议, 以分析 polysome 分数。
Protocol
所有动物的治疗和组织的处理获得的研究是根据协议的机构动物护理和使用委员会在德州理工大学健康科学中心按照国家研究所健康动物福利准则, 96005 号议定书。请牺牲脊椎动物, 根据动物保育和使用委员会的指导方针准备组织。如果缺乏这样一个委员会, 请参考国家卫生研究院动物福利指南。成人 (> 60 天) C57BL/6 小鼠使用。所有动物和组织都是根据美国德州理工大学卫生科学中心的机构动物护?…
Representative Results
在本研究中, 我们描述了 polysomal 分析技术在三种不同来源的应用: 寄生性的利什曼原虫少校、培养的人体细胞和小鼠睾丸。利什曼原虫细胞在悬浮液培养基中自由生长, 培养的人细胞生长在板上的贴膜上, 小鼠睾丸代表组织样本。该方法可以很容易地调整到其他类型的自由生长细胞在悬浮, 不同类型的组织, 或从另一个有机体, 和不同类型的培养细胞。该方法由…
Discussion
蔗糖梯度结合 RNA 和蛋白质分析 Polysome 分馏是分析个体基因或整个 translatome 的平移状态以及调节平移的蛋白质因子作用的有力方法。机械在正常的生理或疾病状态。Polysomal 分析是一种特别适合于研究生物体中的平移调节的技术, 如 trypanosomatids 包括利什曼原虫, 其中转录控制很大程度上缺乏, 基因表达调控主要发生在翻译过程中。
在这里, 我们描述了一个 polysome 分馏协?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者感谢清李的录音帮助。这项研究得到了来自德州理工大学健康科学中心的启动基金的支持, 以及由翻译神经科学和治疗中心 (CTNT) 授予 PN-CTNT 2017-05 AKHRJDHW A.L.K.;部分由 NIH 授予 R01AI099380 对 K.Z. 詹姆斯 c. 霍夫曼和克里斯汀 r 巴卡是喜赛 (干细胞教育 & 研究的整合中心) 学者并且由节目支持。
Materials
| Instruments: | ||
| Gradient master | Biocomp Instruments Inc. | 108 |
| Piston Gradient Fractionator | Biocomp Instruments Inc. | 152 |
| Fraction collector | Gilson, Inc. | FC203B |
| NanoDrop One | Thermo Scientific | NanoDrop One |
| Nikon inverted microscope | Nikon | ECLIPSE Ts2-FL/Ts2 |
| 2720 Thermal Cycler | Applied Biosystems by Life Technologies | 4359659 |
| CO2 incubator | Panasonic Healthcare Co. | MCO-170A1CUV |
| HERATHERM incubator | Thermo Scientific | 51028063 |
| Biological Safety Cabinet, class II, type A2 | NuAire Inc. | NU-543-400 |
| Revco freezer | Revco Technologies | ULT1386-5-D35 |
| Beckman L8-M Ultracentifuge | Beckman Coulter | L8M-70 |
| Centrifuge | Eppendorf | 5810R |
| Centrifuge | Eppendorf | 5424 |
| Ultracentrifuge Rotor SW41 | Beckman Coulter | 331362 |
| Swing-bucket rotor | Eppendorf | A-4-62 |
| Fixed angle rotor | Eppendorf | F-45-30-11 |
| Quant Studio 12K Flex Real-Time PCR machine 285880228 | Applied Biosystems by life technologies | 4470661 |
| TC20 Automated cell counter | Bio-Rad | 145-0102 |
| Hemacytometer | Hausser Scientific | 02-671-51B |
| Software | ||
| Triax software | Biocomp Instruments Inc. | |
| Materials: | ||
| Counting slides, dual chamber for cell counter | Bio-Rad | 145-0011 |
| 1.5 mL microcentrifuge tube | USA Scientific | 1615-5500 |
| Open-top polyclear centrifuge tubes, (14 mm x 89 mm) | Seton Scientific | 7030 |
| Syringe, 5 mL | BD | 309646 |
| BD Syringe 3 mL23 Gauge 1 Inch Needle | BD | 10020439 |
| Nunclon Delta Surface plate, 14 cm | Thermo Scientific | 168381 |
| Nunclon Delta Surface plate, 9 cm | Thermo Scientific | 172931 |
| Nalgene rapid-flow 90mm filter unit, 500 mL, 0.2 aPES | Thermo Scientific | 569-0020 |
| BioLite 75 cm3 flasks | Thermo Scientific | 130193 |
| Nunc 50 mL conical centrifuge tubes | Thermo Scientific | 339653 |
| Chemicals: | ||
| Trizol LS | Ambion by Life Technologies | 10296028 |
| HEPES | Fisher Scientific | BP310-500 |
| Trizma base | Sigma | T1378-5KG |
| Dulbecco's Modified Eagle's Medium-high glucose (DMEM) | Sigma | D6429-500ML |
| Fetal Bovine Serum (FBS) | Sigma | F0926-50ML |
| Penicillin-Streptomycin (P/S) | Sigma | P0781-100ML |
| Lipofectamine 2000 | Invitrogen | 11668-019 |
| Dulbecco's phosphate buffered saline (DPBS) | Sigma | D8537-500ML |
| Magnesium chloride hexahydrate (MgCl2x6H2O) | Acros Organics | AC413415000 |
| Potassium Chloride (KCl) | Sigma | P9541-500G |
| Nonidet P 40 (NP-40) | Fluka (Sigma-Aldrich) | 74385 |
| Recombinant Rnasin Ribonuclease Inhibitor | Promega | N2511 |
| Heparin sodium salt | Sigma | H3993-1MU |
| cOmplete Mini EDTA-free protease inhibitors | Roche Diagnostics | 11836170001 |
| Glycogen | Thermo Scientific | R0551 |
| Water | Sigma | W4502-1L |
| Cycloheximide | Sigma | C7698-1G |
| Chloroform | Fisher Scientific | 194002 |
| Dithiotreitol (DTT) | Fisher Scientific | BP172-5 |
| Ethidium Bromide | Fisher Scientific | BP-1302-10 |
| Ethylenediaminetetraacetic acid disodium dehydrate (EDTA) | Fisher Scientific | S316-212 |
| Optimem | Life Technologies | 22600050 |
| Puromycin dihydrochloride | Sigma | P8833-100MG |
| Sucrose | Fisher Scientific | S5-3KG |
| Trypsin-EDTA solution | Sigma | T4049-100ML |
| Hgh Capacity cDNA Reverse Transcriptase Kit | Applied Biosystems by life technologies | 4368814 |
| Power SYBR Green PCR Master Mix | Applied Biosystems by life technologies | 4367659 |
| HCl | Fisher Scientific | A144SI-212 |
| Isopropanol | Fisher Scientific | BP26324 |
| Potassium Hydroxide (KOH) | Sigma | 221473-500G |
| Anti-RPL11 antibody | Abcam | ab79352 |
| Ribosomal protein S6 (C-8) antibody | Santa Cruz Biotechnology Inc. | sc-74459 |
| 1xM199 | Sigma | M0393-10X1L |
| Lithium cloride | Sigma | L-9650 |
| Dimethyl sulfoxide (DMSO) | Fisher Scientific | D128-500 |
| Gel Loading Buffer II | Thermo Scientific | AM8546G |
| UltraPure Agarose | Thermo Scientific | 16500-100 |
| Trichloracetic acid (TCA) | Fisher Scientific | A322-100 |
| SuperSignal West Pico PLUS chemiluminescent substrate | Thermo Scientific | 34580 |
| Formaldehyde | Fisher Scientific | BP531-500 |
| Sodium Dodecyl Sulfate (SDS) | Sigma | L5750-1KG |
| Phenylmethylsulfonyl fluoride (PMSF) | Sigma | P7626-5G |
| RNeasy Mini kit | Qiagen | 74104 |
| Adenosine 5′-triphosphate disodium salt hydrate (ATP) | Sigma | A1852-1VL |
| Cytosine 5'-triphosphate disodium salt hydrate (CTP) | Sigma | C1506-250MG |
| Uridine 5'-triphosphate trisodium salt hydrate (UTP) | Sigma | U6625-100MG |
| Guanosine 5'-triphosphate sodium salt hydrate (GTP) | Sigma | G8877-250MG |
| SP6 RNA Polymerase | NEB | M0207S |
| Pyrophoshatase | Sigma | I1643-500UN |
| Spermidine | Sigma | S0266-1G |
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Citar este artículo
Karamysheva, Z. N., Tikhonova, E. B., Grozdanov, P. N., Huffman, J. C., Baca, K. R., Karamyshev, A., Denison, R. B., MacDonald, C. C., Zhang, K., Karamyshev, A. L. Polysome Profiling in Leishmania, Human Cells and Mouse Testis. J. Vis. Exp. (134), e57600, doi:10.3791/57600 (2018).