利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白
1College of Chemistry and Molecular Engineering,Peking University, 2Beijing National Laboratory for Molecular Sciences,Peking University, 3Peking-Tsinghua Center for Life Sciences,Peking University, 4Synthetic and Functional Biomolecules Center,Peking University, 5Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education,Peking University
Summary
本文介绍了应用单击化学辅助 RNA-路特捕获 (无花果) 策略来识别与编码和非编码 rna 结合的蛋白质的详细协议。
Abstract
RNA 结合蛋白 (限制性商业惯例) 的综合鉴定是了解细胞转录后调控网络的关键。一种广泛使用的策略, 用于限制性捕获利用酸化 [聚 (a)] 的靶 rna, 这主要发生在真核成熟 mRNAs, 留下大多数非聚 (A) rna 的结合蛋白不明。在这里, 我们描述了一个最近报告的方法称为单击化学辅助 RNA-路特捕获 (无花果) 的详细过程, 这使转录组范围捕获的聚 (a) 和非聚 (a) 限制性商业惯例的代谢标签结合rna,体内UV 交联, 和叠标记。
Introduction
人类基因组转录成各种类型的编码和非编码 rna (非编码 rna), 包括 mRNAs、rRNAs、tRNAs、小核 rna (snRNAs)、小核仁 rna (snoRNAs) 和长非编码 rna (lncRNAs)1。大多数 rna 具有限制性商业惯例的服装和功能作为核糖颗粒 (RNPs)2。因此, 限制性商业惯例的综合识别是了解 rna 和限制性商业惯例之间的调控网络的先决条件, 这是涉及各种人类疾病3,4,5。
过去几年见证了限制性商业惯例在各种真核系统2,6, 包括人类7,8,9,10,11中发现的巨大的促进作用,鼠标12,13,14, 酵母9,15,16, 斑马鱼17,果蝇18,19,线虫线虫16,拟南芥20,21,22, 和人类寄生虫23,24,25.这些进步是由城堡等开发的一个限制性的捕获策略促进。7和巴尔茨等。8在 2012年, 它结合了在体内UV 交联的 RNA 及其相互作用的蛋白质, 寡核苷酸 (dT) 捕获聚 (A) rna 和质谱 (MS) 基于蛋白质组分析。然而, 鉴于聚 (a) 多数存在于成熟的 mRNAs 上, 它仅占真核转录组26的 3%-5%, 这种广泛使用的策略无法捕获与非聚 (A) rna 的限制性商业惯例交互, 包括大多数非编码 rna和预 mRNAs。
在这里, 我们报告了一个最近制定的战略的详细程序, 以转录组全捕获的聚 (a) 和非聚 (a) 限制性商业惯例27。被称为无花果, 这种策略结合体内UV 交联和 rna 代谢标记与光敏和 “可点击的” 核苷类似物 (其中包含一个叠功能组, 可以参与点击反应), 4-thiouridine (4SU) 和 5-ethynyluridine (EU)。通过无花果策略获得理想结果的关键步骤是有效的代谢标记、UV 交联和点击反应以及 RNA 完整性的维护。因为 cu (i) 作为催化剂在单击反应可能导致 rna 碎片, 一个 Cu (i) 配体, 可以减少 RNA 碎片是必不可少的。我们描述如何在细胞裂解液中执行高效的单击反应, 而不会导致严重的 RNA 降解。
虽然在 HeLa 细胞中的限制性捕获和鉴定仅在本协议中描述, 无花果策略可应用于各种细胞类型和可能的生物体。除了限制性捕获, 该协议还提供了简化的分步过程, 用于 MS 样品制备和蛋白质鉴定和定量, 这对于那些不熟悉蛋白质组实验的人是有帮助的。
Protocol
注意: 在适用的情况下, 使用的试剂应以无 rna 的形式购买, 或在无核糖核酸酶中溶解, 溶剂 (大部分为酯二乙酯 (DEPC) 处理的水)。在处理 RNA 样品和无核糖核酸酶试剂时, 务必戴上手套和口罩, 并经常更换, 以避免核糖核酸酶污染。 1. 代谢标记和 UV 交联细胞裂解物的制备 欧盟和4SU 的代谢合并 杜尔贝科的改良鹰培养基 (DMEM) 中培养的 HeLa 细胞, 辅以10% ?…
Representative Results
提出了质量控制步骤的代表性结果。结果包括步骤2.3.2 中所述的凝胶荧光分析的数字 (图 1)、步骤4.1.3 中描述的西方印迹分析 (图 2A) 和步骤4.2.2 中描述的银染色分析 (图 2B)。质量控制步骤对于优化无花果协议至关重要。在大规模的限制性鉴定实验的制备中始终包括质量控制。 <p class="jove_content" fo:keep-tog…
Discussion
保持公平 RNA 完整性是成功无花果实验的关键之一。随着 Cu (I) 的适当配体和仔细操作, RNA 降解可以大大减少, 虽然部分降解被观察到。在实验样品中, 欧盟和4SU 的替代比分别为1.18% 和 0.46% (未显示数据)。对于长度为 2000 nt 的完整 rna, 约90% 的 rna 包含至少一个欧盟和一个4SU。对于部分退化的 rna, 长度为 1000 nt, 约70% 的 rna 包含至少一个欧盟和一个4SU。因此, rna 的部分降解不会显著降低无花果的效率, ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由国家自然科学基金资助91753206、21425204和21521003和国家重点研究开发项目2016YFA0501500。
Materials
| HeLa | ATCC | ||
| DMEM (Dulbecco's Modified Eagle Medium) | Thermo Fisher Scientific | 11995065 | |
| FBS (Fetal Bovine Serum) | Thermo Fisher Scientific | 10099141 | |
| Penicillin & Streptomycin | Thermo Fisher Scientific | 15140122 | |
| EU (5-ethynyl uridine) | Wuhu Huaren Co. | CAS:69075-42-9 | |
| 4SU (4-thiouridine) | Sigma Aldrich | T4509 | |
| 10×PBS (Phosphate-Buffered Saline) | Thermo Fisher Scientific | AM9625 | |
| UV cross-linker | UVP | CL-1000 | Equiped with 365-nm UV lamp |
| DEPC (Diethyl pyrocarbonate) | Sigma Aldrich | D5758 | To treat water. Highly toxic! |
| Tris·HCl, pH 7.5 | Thermo Fisher Scientific | 15567027 | |
| LiCl | Sigma Aldrich | 62476 | |
| Nonidet P-40 | Biodee | 74385 | |
| EDTA-free protease inhibitor cocktail | Thermo Fisher Scientific | 88265 | One tablet for 50 mL lysis buffer. |
| LDS (Lithium dodecyl sulfate) | Sigma Aldrich | L9781 | |
| 15-mL ultrafiltration tube (10 kDa cutoff) | Millipore | UFC901024 | |
| 0.5-mL ultrafiltration tube (10 kDa cutoff) | Millipore | UFC501096 | |
| Streptavidin magnetic beads | Thermo Fisher Scientific | 88816 | |
| DMSO (Dimethyl sulfoxide) | Sigma Aldrich | 41639 | |
| Azide-biotin | Click Chemistry Tools | AZ104 | |
| Copper(II) sulfate | Sigma Aldrich | C1297 | |
| THPTA [Tris(3-hydroxypropyltriazolylmethyl)amine] | Sigma Aldrich | 762342 | |
| Sodium ascorbate | Sigma Aldrich | 11140 | |
| Azide-Cy5 | Click Chemistry Tools | AZ118 | |
| LDS sample buffer (4×) | Thermo Fisher Scientific | NP0008 | |
| 10% bis-Tris gel | Thermo Fisher Scientific | NP0301BOX | |
| EDTA | Thermo Fisher Scientific | AM9260G | |
| RNase A | Sigma Aldrich | R6513 | |
| SDS (Sodium dodecyl sulfate) | Thermo Fisher Scientific | 15525017 | |
| NaCl | Sigma Aldrich | S3014 | |
| Brij-97 [Polyoxyethylene (20) oleyl ether] | J&K | 315442 | |
| Triethanolamine | Sigma Aldrich | V900257 | |
| Streptavidin agarose | Thermo Fisher Scientific | 20353 | |
| Urea | Sigma Aldrich | U5378 | |
| Sarkosyl (N-Lauroylsarcosine sodium salt) | Sigma Aldrich | 61743 | |
| Biotin | Sigma Aldrich | B4501 | |
| Sodium deoxycholate | Sigma Aldrich | 30970 | |
| MaxQuant | Version: 1.5.5.1 |
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Cite This Article
Huang, R., Han, M., Meng, L., Chen, X. Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy. J. Vis. Exp. (140), e58580, doi:10.3791/58580 (2018).