Name: monitoring protein-rna interaction dynamics in vivo at high temporal resolution using χcrac
Uploaded: 2020-05-09T13:00:00.000+00:00
Duration: 9 min 15 s
Description: Watch this Scientific Journal Video about Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using Ï‡CRAC at JoVE.com

这项工作得到了威康信托基金会（091549给S.G.和109093/Z/15 / A给S.M.），威康信托基金会细胞生物学中心核心资助（092076）和医学研究委员会非临床高级研究奖学金（MR / R008205 / 1到S.G.），欧洲分子生物学组织的长期博士后奖学金（ALTF 1070-2017到R.A.C.）的资助， 以及丹麦独立研究基金（T.H.J）。

A. Langford和W. Worboys隶属于商业公司UVO3。他们在研究设计、数据收集和解释或提交作品发表的决定中没有任何作用。

χCRAC方法与新的交联和细胞收获装置相结合，具有巨大的潜力，因为它适用于广泛的模式生物，因此应该引起RNA领域的普遍兴趣。χCRAC 可用于许多领域。例如，该方法可用于测量蛋白质成大分子复合物的分层组装，例如剪接体和核糖体，这通常涉及蛋白质和RNA分子之间的动态相互作用。我们现在还经常使用它来监测当细胞受到各种压力时RNA衰变因子与其底物之间的相互作用。这使我们能够确定这些因素在适应性反应的哪个阶段最活跃，它们与哪些底物结合，以及这些相互作用的动态性。这些数据应使研究人员能够确定每个因素在适应环境变化方面的相对贡献。
χCRAC 使用双亲和纯化标签（HTF 或 HTP）在高度严格和变性的条件下纯化蛋白质。这确保了共纯化的RNA对于与目标蛋白共价交联的RNA具有高度富集性。但是，依赖亲和力标记有缺点。例如，标签可能会干扰蛋白质功能，这可能会使其RNA结合相互作用组的读数失真。此外，对于某些模式生物，可能并不总是能够利用标签，因为尚未获得将DNA片段整合到基因组中或转化表达质粒的遗传工具。然而，改变χCRAC方案的某些部分以使其与依赖抗体纯化RBP的基于CLIP的方案兼容是很简单的。事实上，这项研究表明，可以将基于iCLIP的纯化与我们的交联剂相结合。我们现在正在开发CLIP协议，以研究人类RNA结合蛋白与新生RNA转录本的时间关联。
当对新蛋白质进行χCRAC时，必须优化紫外线照射以诱导最大的交联。这一点很重要，因为高紫外线照射会降低纯化步骤中RNA的回收率。表达重组RBP的细胞暴露于各种紫外线剂量，100 mJ / cm 2，250 mJ / cm 2，500 mJ / cm 2和1 J/ cm 2。然后捕获RNP，并对RNA片段化和放射性标记。之后，通过SDS-PAGE解析RNP，并进行放射自显影，以推断哪种暴露产生最强烈的信号（即最大交联）。
一旦实验条件得到优化，建议在进行χCRAC时进行几个对照实验。首先，可以使用紫外线照射的未标记样品来监测与纯化微球的背景结合。其次，当在转移实验期间应用χCRAC时，细胞移回原始培养基的第二个时间序列可以研究细胞本身的过滤是否诱导RNA水平或蛋白质 - RNA相互作用的变化。
如引言所述，最近发表的许多论文提出了对CLIP协议的一些优化。这包括使用荧光标记的适配器通过红外扫描10 检测蛋白质-RNA复合物，以及对各种核酸纯化和大小选择步骤的优化，以增加所得文库的复杂性12，45。我们目前正在实施其中一些改进，以进一步完善χCRAC协议。这里介绍的协议已经包含对原始CRAC和χCRAC协议的许多改进，这些改进增加了数据的复杂性。例如，以前，在分离SDS-PAGE凝胶上的交联放射性蛋白质-RNA复合物后，将它们转移到硝酸纤维素膜上，并从印迹中分离交联RNA。然而，RNP的转移和随后的RNA提取可能非常低效，特别是在处理大RBP如RNA聚合酶亚基时。这可能导致交联RNA的回收率显着降低。在当前方案中，交联RNA直接从SDS-PAGE凝胶切片中提取，如图 1所示。这增加了交联RNA的回收率。此外，在cDNA的PCR扩增后，产物最初在3%的低熔解温度琼脂糖凝胶上分离，然后从凝胶中提取175-300 bp PCR产物。然而，这些凝胶很容易过载，导致DNA分离非常差。用预制TBE凝胶代替琼脂糖凝胶可实现更一致的尺寸分离和更好的PCR产物回收率。

An erratum was issued for:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/t/61027">Monitoring Protein-RNA Interaction Dynamics in vivo at High Temporal Resolution using &#967;CRAC</a>. The Authors section was updated from:
Stuart W. McKellar1 
Ivayla Ivanova1 
Robert W. van Nues2 
Ross A. Cordiner3 
Will Worboys4 
Andrew Langford4 
Torben Heick Jensen3 
Sander Granneman1 
1Centre for Synthetic and Systems Biology, University of Edinburgh 
2Institute of Cell Biology, University of Edinburgh 
3Department of Molecular Biology and Genetics, Aarhus University, 4UVO3 Ltd.
to:
Stuart W. McKellar1 
Ivayla Ivanova1 
Robert W. van Nues2 
Ross A. Cordiner3 
Mehak Chauhan1 
Niki&#160;Christopoulou1 
Will Worboys4 
Andrew Langford4 
Torben Heick Jensen3 
Sander Granneman1 
1Centre for Engineering Biology, University of Edinburgh 
2Institute of Cell Biology, University of Edinburgh 
3Department of Molecular Biology and Genetics, Aarhus University, 4UVO3 Ltd.

An erratum was issued for:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/t/61027">Monitoring Protein-RNA Interaction Dynamics in vivo at High Temporal Resolution using &#967;CRAC</a>. The Authors section was updated.

Erratum: Monitoring Protein-RNA Interaction Dynamics in vivo at High Temporal Resolution using &#967;CRAC

RNA通常依靠RBP来发挥其功能，这引起了人们对理解这些分子之间动力学的极大兴趣。已在多种生物体中鉴定出许多RBP。然而，在体内研究RBP-RNA相互作用一直是出了名的困难。研究这种相互作用的一个重大突破是CLIP1的出现。该方法利用紫外（UV，254 nm）照射诱导RBP与其直接结合的RNA之间的共价键（即零距离交联）。随后，在严格的条件下对感兴趣的RBP进行免疫纯化，以确保仅鉴定与蛋白质共价交联的RNA。结合的RNA然后用RNA酶部分消化，随后转化为cDNA文库进行测序。高纯化严格性非常重要，因为它大大提高了蛋白质和RNA回收率的特异性，通过交联核糖核蛋白（RNP）复合物的SDS-PAGE纯化也进一步增强了特异性。CLIP和相关方法还提供了对蛋白质结合位点的核苷酸分辨率洞察，因为在测序文库的制备过程中，与RNA交联的氨基酸经常终止逆转录酶或导致酶在该位点引入突变1，2，3。
自推出以来，最初的CLIP协议已经产生了各种各样的衍生方法。一个特别重要的突破来自HITS-CLIP（或CLIP-seq）的开发，它将高通量测序与CLIP方法3相结合。此后，所有基于CLIP的方法都采用了这种方法。iCLIP引入了RNase介导的修剪和衔接连接技术的改进，有助于更准确地映射RBP结合位点4。PAR-CLIP 将 4 硫代尿苷/尿嘧啶标记与 365 nm 的交联相结合，使得通过分析 T-C 取代物5 来绘制交联位点成为可能。CRAC、尿素-iCLIP、dCLIP和uvCLAP引入了变性条件和双重亲和纯化步骤，进一步减少了与亲和树脂的背景结合，并进一步提高了蛋白质捕获的特异性2，6，7，8，9。此外，CRAC、uvCLAP 和 dCLIP 引入了用亲和标签标记感兴趣的 RBP，从而克服了产生特异性抗体的需要。
还进行了一些优化以加快 CLIP 方法。最初的CLIP协议利用捕获的RNA的放射性标记，以便在SDS-PAGE之后可视化RBP-RNA复合物。然而，对于没有为此类工作设立的实验室来说，使用放射性可能会有问题。irCLIP包含一个荧光团偶联接头，可通过红外成像10 进行可视化，sCLIP利用捕获的RNA的生物素化，以便通过链霉亲和素偶联的HRP11对其进行可视化。此外，eCLIP完全放弃了RNA标记;相反，仅根据其已知的大小12切除蛋白质。基于链霉亲和素的纯化也已用于加速FAST-iCLIP中的文库制备过程，其中生物素化的3'接头连接到RNA上，并用于在逆转录和环化后实现纯化13。iCLIP协议的其他增强功能也大大增加了库的复杂性4.
最后，对CLIP进行了修改，以便能够从不同的细胞亚区室14，15捕获RBP，使用可光活化核糖核苷5，16，17的脉冲诱导可视化新转录的RNA，捕获甲基化RNA18，19，20，检查RNA-RNA相互作用21，22，并映射3'末端23，24.
尽管基于CLIP的技术在帮助我们理解RBP和RNA之间的相互作用方面做出了巨大贡献，但它一直受到UV交联效率低下的限制。尽管在单层中生长的培养细胞通常相对容易交联，但这在组织或溶液中的细胞中更具挑战性。组织可能需要多轮紫外线照射才能渗透到所需的细胞层，而微生物细胞通常在含有芳香族紫外线吸收化合物的丰富培养基中生长25。事实上，长达 30 分钟的紫外线照射时间已被用于在此类样品的 RBP 与其结合的 RNA 之间产生足够的交联26，27，28。这种延长的紫外线照射会在细胞内诱导应激反应，例如紫外线诱导的DNA损伤，这可能会污染某些应用中的最终数据。
大多数CLIP研究都集中在生成细胞中特定蛋白质 - RNA相互作用的单个"快照"。然而，蛋白质-RNA相互作用本质上是动态的，特别是当细胞受到环境变化的影响时。这可能包括必需营养素的可用性突然减少或温度的快速变化。因此，要真正了解RBP在压力下的作用，最好进行时间分辨分析，因为它们可以捕获压力期间RBP目标的全部范围，并确定所选RBP在压力反应的哪个阶段处于活动状态。特别是，对酵母的研究表明，适应的最初几分钟对生存绝对至关重要，细菌中的RNA半衰期可以从几分钟到几秒钟不等29，30，31，32，33。因此，理想情况下，这种时间分辨分析应该在高时间分辨率下进行。然而，较长的交联时间使得早期适应性反应的研究特别具有挑战性。
为了克服这些问题，我们最近开发了一种改进的方法，能够在长达一分钟的时间尺度上交联和收获细胞。我们的χCRAC方法允许以以前未见过的分辨率定量测量RBP-RNA相互作用的动态变化。该方法的关键是开发了一种新型紫外线照射装置32 ，该装置将酵母和溶液中细菌中所需的交联时间减少了约10倍，有效地瞬间冷冻RBP-RNA相互作用。此外，为了在紫外线照射后快速收获细胞，我们开发了一种真空过滤装置，可以在大约 30 秒32 的 0.5 L 培养物中收获指数生长的酵母。这些技术创新允许以分钟级分辨率研究RBP-RNA动力学。此外，我们还对原始CRAC协议2 进行了一些优化，以提高其实用性。
使用χCRAC，我们最近研究了酵母核RBP，Nab3的靶组，以响应葡萄糖剥夺。在 酿酒酵母中，Nab3可以与Nrd1，RBP和RNA解旋酶Sen1形成复合物，形成NNS复合物。NNS与RNA聚合酶和新生转录本结合可以触发转录终止34。这种复合物主要参与去除神秘的非编码RNA转录本，但也已被证明可以调节蛋白质编码基因的表达。该研究显示，Nab3在仅一分钟的应激后就对非编码和编码转录本的靶向不同32。我们证明了Nab3的共转录终止导致逆转录转座子基因的非常短暂的脉冲样表达，这很难使用传统的基于CLIP的方法检测到。此外，我们的紫外交联剂中较短的紫外照射时间也显著提高了短寿命非编码RNA的回收率32。χCRAC可能是一个关键工具，不仅可以阐明RBP如何在即时时间尺度上塑造对压力的反应，还可以阐明它们在响应的整个生命周期中不断变化的角色。这份手稿详细概述了χCRAC协议中的所有步骤。出于说明目的，该方法用于研究参与转录终止和RNA衰变35，36的酵母Nrd1蛋白及其RNA靶蛋白在多个时间点响应葡萄糖剥夺。最后，我们还证明，我们的紫外线照射单元可以快速将RBP与HeLa细胞中的RNA交联，从而可以在贴壁细胞中进行高分辨率的时间分辨分析。

<table><tbody><tr><td>1,4-dithioreitol</td><td>Merck</td><td>10708984001</td><td>Buffer component in mammalian cell lysis</td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf</td><td>0030 120.086</td><td>General reaction tube</td></tr><tr><td>2 mL tubes</td><td>Eppendorf</td><td>0030 123.344</td><td>For holding columns and collection of waste</td></tr><tr><td>32P-yATP</td><td>Perkin Elmer</td><td>NEG502Z-250</td><td>For radiolabelling the 5&#039; end of the RNA</td></tr><tr><td>4-12% Bis-Tris gel</td><td>Invitrogen</td><td>NP0321BOX</td><td>SDS-PAGE gel</td></tr><tr><td>4X loading buffer</td><td>Novex</td><td>NP0008</td><td>Protein loading dye concentrate</td></tr><tr><td>50 bp ladder</td><td>New England Biolabs</td><td>N3236</td><td>Reference ladder for excising region of interest from the amplified cDNA library</td></tr><tr><td>50% PEG</td><td>NEB</td><td>B100045</td><td>For the L5 linker ligation</td></tr><tr><td>6% TBE gel</td><td>Invitrogen</td><td>EC6265BOX</td><td>For separation and purification of the cDNA library</td></tr><tr><td>Acetone</td><td>ACROS Organics</td><td>423245000</td><td>Washing of TCA-precipitated proteins</td></tr><tr><td>anti-FLAG beads</td><td>Sigma Aldrich</td><td>M8823-1ML</td><td>For purifcation of FLAG-tagged RBPs</td></tr><tr><td>ATP (100 mM)</td><td>Thermo Fisher Scientific</td><td>R0441</td><td>For ligation of the L5 linker onto the 5&#039; end of captured RNAs</td></tr><tr><td>Beta-mercaptoethanol</td><td>Sigma Aldrich</td><td>M3148-100ML</td><td>Buffer component</td></tr><tr><td>Biomax MS intensifying screen</td><td>Sigma Aldrich</td><td>Z363162-1EA</td><td>For intensifying the autoradiogram signal</td></tr><tr><td>Chloroform</td><td>Thermo Fisher Scientific</td><td>1010219</td><td>For phenol-chloroform extraction following RNA purification</td></tr><tr><td>cOmplete EDTA-free protease inhibitor cocktail</td><td>Roche</td><td>11873580001</td><td>For inhibition of cellular proteases after lysis</td></tr><tr><td>Complete supplement mixture -TRP</td><td>Formedium</td><td>DCS0149</td><td>For preparation of synthetic defined medium</td></tr><tr><td>Costar Spin-X 0.22 &amp;micro;m filters</td><td>Sigma Aldrich</td><td>CLS8160</td><td>For isolating the excised cDNAs following gel extraction</td></tr><tr><td>DNase RQ1</td><td>Promega</td><td>M6101</td><td>For DNA digest following cell lysis</td></tr><tr><td>dNTPs (10 mM)</td><td>Sigma Aldrich</td><td>4638956001</td><td>For reverse transcription and PCR</td></tr><tr><td>Ethanol</td><td>Thermo Fisher Scientific</td><td>10041814</td><td>For phenol-chloroform extraction following RNA purification and DNA precipitation</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Invitrogen</td><td>AM9261</td><td>For protease K buffer</td></tr><tr><td>Exonuclease I</td><td>New England Biolabs</td><td>M0293</td><td>For degradation of primers following PCR</td></tr><tr><td>Glass microfiber filters</td><td>Whatman</td><td>1823-010</td><td>For isolating the excised cDNAs following gel extraction</td></tr><tr><td>Glucose</td><td>Formedium</td><td>GLU03</td><td>For preparation of glucose-containing, synthetic defined medium</td></tr><tr><td>Glycogen (20 mg/mL)</td><td>Roche</td><td>10901393001</td><td>Precipitation of proteins, RNA and DNA</td></tr><tr><td>GST-TEV</td><td>Homemade</td><td></td><td>Construct and purification protocol is available upon request</td></tr><tr><td>Guanidium hydrochloroide</td><td>Thermo Fisher Scientific</td><td>10071503</td><td>Required for pulldown denaturing conditions and washing buffer</td></tr><tr><td>IgG beads</td><td>GE Healthcare</td><td>17-0969-01</td><td>For purification of protein A-tagged RBPs</td></tr><tr><td>Imidazole</td><td>Sigma Aldrich</td><td>I2399-100G</td><td>For elution of captured proteins from Nickel beads</td></tr><tr><td>Isoamyl alcohol</td><td>Thermo Fisher Scientific</td><td>A393-500</td><td>For phenol-chloroform extraction following RNA purification</td></tr><tr><td>Luna Universal One-Step RT-qPCR</td><td>NEB</td><td>E3005S</td><td>For qPCR of the cDNA in order to calculate required number of PCR cycles</td></tr><tr><td>Magnesium chloride</td><td>Fluka Analytical</td><td>63020-1L</td><td>For PNK buffer</td></tr><tr><td>Membrane filters</td><td>Millipore</td><td>AAWP09000 for yeast or HAWP09000 for bacteria</td><td>For vacuum filtration of cells</td></tr><tr><td>Micro bio-spin columns</td><td>Biorad</td><td>732-6204</td><td>For collecting eluate after gel extraction</td></tr><tr><td>Ni-NTA beads</td><td>Qiagen</td><td>30210</td><td>For secondary protein capture</td></tr><tr><td>NP-40</td><td>Sigma Aldrich</td><td>I8896-100ML</td><td>Buffer component</td></tr><tr><td>Pfu polymerase</td><td>Promega</td><td>M7741</td><td>For amplification of the cDNA library</td></tr><tr><td>Phenol</td><td>Sigma Aldrich</td><td>P4682-400ML</td><td>For phenol-chloroform extraction following RNA purification</td></tr><tr><td>Pierce spin columns</td><td>Thermo Fisher Scientific</td><td>69725</td><td>For on-column enzymatic reactions</td></tr><tr><td>Protease K</td><td>Roche</td><td>3115887001</td><td>For degradation of the RBP following gel extraction</td></tr><tr><td>Quartz Petri dish</td><td>UVO3</td><td>N/A</td><td>For cross-linking of adherent cells. Available from https://www.vari-x-link.com for 400 GBP</td></tr><tr><td>Radiography films</td><td>Amersham</td><td>28906843</td><td>For autoradiography visualisation</td></tr><tr><td>RNAClean XP beads</td><td>Beckmann</td><td>A63987</td><td>SPRI beads for clean up of RNAs and cDNAs</td></tr><tr><td>RNase H</td><td>New England Biolabs</td><td>M0297</td><td>For degradation of RNAs following reverse transcription</td></tr><tr><td>RNase-It</td><td>Agilent</td><td>400720</td><td>For RNA digestion</td></tr><tr><td>rRNasin</td><td>Promega</td><td>N2511</td><td>For inhibition of any contaminating RNases during enzymatic reaction</td></tr><tr><td>Sodium acetate</td><td>Sigma Aldrich</td><td>S2889-1KG</td><td>For phenol-chloroform extraction following RNA purification and DNA precipitation</td></tr><tr><td>Sodium chloride</td><td>Thermo Fisher Scientific</td><td>7647-14-5</td><td>Buffer component</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma Aldrich</td><td>D6750-100G</td><td>Buffer component in mammalian cell lysis</td></tr><tr><td>Sodium dodecylsulfate</td><td>Sigma Aldrich</td><td>L3771-1KG</td><td>For protease K buffer</td></tr><tr><td>SUPERase-In</td><td>Invitrogen</td><td>AM2694</td><td>For inhibition of cellular RNases after lysis</td></tr><tr><td>SuperScript IV</td><td>Thermo Fisher Scientific</td><td>18090010</td><td>For reverse transcription</td></tr><tr><td>T4 PNK</td><td>New England Biolabs</td><td>M0201</td><td>For radiolabelling the 5&#039; end of the RNA</td></tr><tr><td>T4 RNA ligase 1</td><td>New England Biolabs</td><td>M0204</td><td>For ligation of the L5 adaptor onto the RNA 5&#039; end</td></tr><tr><td>T4 RNase ligase 2, truncated K222Q</td><td>NEB</td><td>M0351S</td><td>For ligation of the App_PE linker onto the 3&#039; end of captured RNAs</td></tr><tr><td>TBE buffer (10X)</td><td>Invitrogen</td><td>15581-028</td><td>For running TBE gels</td></tr><tr><td>TEV protease</td><td>Homemade</td><td></td><td>For eluting captured proteins following FLAG capture</td></tr><tr><td>Thermosensitive alkaline phosphatase</td><td>Promega</td><td>M9910</td><td>For 5&#039; and 3&#039; dephosphorylation of RNAs</td></tr><tr><td>Trichloroacetic acid (100%)</td><td>Sigma Aldrich</td><td>T0699-100ML</td><td>For precipitation of RBP-RNA complexes</td></tr><tr><td>Tris hydrochloride</td><td>Invitrogen</td><td>15504-020</td><td>Buffer component</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787-100ML</td><td>Buffer component in mammalian cell lysis</td></tr><tr><td>Vari Filter</td><td>UVO3</td><td>N/A</td><td>Device for vacuum harvesting cells. Available from https://www.vari-x-link.com for 100 GBP</td></tr><tr><td>Vari-X-Linker</td><td>UVO3</td><td>N/A</td><td>Cross-linker for cross-linking cells. Available from https://www.vari-x-link.com for 16,000 GBP</td></tr><tr><td>Yeast nitrogen base</td><td>Formedium</td><td>CYN0410</td><td>For preparation of synthetic defined medium</td></tr><tr><td>Zirconia beads</td><td>Thistle</td><td>11079105Z for yeast or 11079101Z for bacteria</td><td>For cell lysis via bead beating</td></tr></tbody></table>

monitoring protein-rna interaction dynamics in vivo at high temporal resolution using &#967;crac

RNA结合蛋白（RBP）与其RNA底物之间的相互作用表现出流动性和复杂性。在其生命周期内，单个RNA可以被许多不同的RBP结合，这些RBP将调节其产生，稳定性，活性和降解。因此，已经做了很多工作来理解这两种分子之间存在的动力学。一个特别重要的突破是"cross-l inink and immunoprecipitation"（CLIP）的出现。该技术允许严格研究哪些RNA与特定的RBP结合。简而言之，将感兴趣的蛋白质在体内与其RNA底物进行紫外交联，在高度严格的条件下纯化，然后将与蛋白质共价交联的RNA转化为cDNA文库并进行测序。自其概念以来，已经开发了许多衍生技术，以使CLIP适合特定的研究领域。然而，众所周知，使用紫外线进行交联效率低下。这导致暴露时间延长，使得RBP-RNA相互作用的时间研究变得不可能。为了克服这个问题，我们最近设计并制造了大大改进的紫外线照射和细胞收集设备。使用这些新工具，我们开发了一种以高时间分辨率对活细胞中的RBP-RNA相互作用进行时间分辨分析的方案:动力学CRoss-link和cDNA的Nalysis（χCRAC）。我们最近使用这种技术来研究酵母RBP在营养应激适应中的作用。本手稿详细介绍了χCRAC方法，并介绍了使用Nrd1 RBP获得的最新结果。

为了证明χCRAC方法的有效性，对表达HTP标记Nrd1蛋白的酵母菌株进行了时程实验。 图1提供了描述该方法工作原理的详细原理图。与Nab3一样，Nrd1参与多种RNA转录本的核RNA衰变37。Corden实验室先前的工作表明，当细胞遭受葡萄糖饥饿时，Nrd1与其RNA靶标的结合会发生显着变化28，38。因此，在含有葡萄糖（SD-TRP）的培养基中呈指数生长的细胞在一段时间内转移到不含葡萄糖（S-TRP）的同一培养基上，以监测Nrd1-RNA相互作用的动态变化。在移位前和1、2、4、8、14和20分钟后，在Vari-X-接头室（图3A）中采集样品并交联。用于细胞生长的培养基故意缺乏色氨酸，以减少这种芳香族氨基酸对紫外线的吸收。请注意，最好使用经过过滤灭菌的合成培养基，因为高压灭菌培养基会导致糖的焦糖化。这会降低交联效率。
图4A 显示了χCRAC实验的代表性放射自显影。请注意，在此示例中，样本未合并在一起。相反，每个都在凝胶上单独运行。建议将其用于初始实验测试，以证明蛋白质在所有测试时间点都有效地与RNA交联。在RBP的预期分子量下观察到特别强烈的信号，表示蛋白质与非常短的放射性标记RNA结合，不适合测序。因此，分离出该条带上方的涂抹信号，即与较长RNA片段交联的蛋白质。该片段是从蛋白质条带上方加上约30 kDa切割的。 图4B 显示了切除后的放射自显影，蛋白质与留在凝胶中的短RNA交联，现在切除了先前的涂片信号。
逆转录后，必须使用PCR扩增cDNA文库。但是，必须避免文库的过度扩增，因为这会偏向由聚合酶优先扩增的序列并产生PCR伪影。过度放大的库还包含大量重复序列，这些序列会浪费序列器上的读取。为了计算扩增最终文库的理想PCR循环次数，使用P5和BC寡核苷酸通过qPCR扩增cDNA的等分试样。选择文库达到峰值荧光的第一个循环作为PCR循环计数。 图4C 给出了来自典型cDNA文库的qPCR示例，其峰循环计数为16。然后将该值用于最终的χCRAC PCR。为了处理测序的数据，我们使用实验室先前开发的软件（pyCRAC）和相应的管道来分析动力学CRAC数据（Nues等人，2017; https://git.ecdf.ed.ac.uk/sgrannem/pycrac，https://bitbucket.org/sgrann/kinetic_crac_pipeline/src/default/）。这些开源软件工具能够对数据进行解复用和修剪，去除PCR重复，鉴定具有统计学意义的峰，将读数聚类为连续序列，并识别结合基序39。有关这些工具如何运作的更多详细信息，请参阅其各自的网页。
我们还开始为哺乳动物细胞开发χCRAC协议。大多数哺乳动物细胞系都是单层生长的，我们带有紫外线渗透袋的交联剂中的托盘不适合贴壁细胞实验。为了克服这个问题，我们开发了一个载物台，用户可以用贴壁细胞对1-2个培养皿（直径150毫米，深度25毫米）进行紫外线照射（图3B）。作为第一个测试，通过使用抗GFP抗体和传统的基于CLIP的纯化交联和捕获稳定标记的GFP-RBM7来测量哺乳动物细胞交联剂的效率。如图 5A所示，交联剂能够使用254nm紫外线照射从单层生长的哺乳动物细胞中回收蛋白质-RNA复合物，其效率与广泛使用的紫外线照射装置相当。然而，通常用于紫外交联实验的标准细胞培养塑料器皿对254nm紫外是不可穿透的。因此，在我们的交联剂中，细胞只会接受来自紫外线灯上岸的照射。为了克服这个问题，我们开发了一种用于细胞生长和交联的紫外线渗透石英培养皿。使用石英培养器皿在短短 2 秒的紫外线照射下显示出蛋白质-RNA 复合物的稳健回收率（图 5B）。当与哺乳动物细胞的RBP捕获方法（如CLIP技术）结合使用时，这些较短的交联时间适合于时间进程，以响应遗传毒性应激或蛋白质因子的快速消耗，或与转录或细胞周期同步并行恢复RBP的时空RNA结合谱。
图 6 显示了 χCRAC 管道处理的 Nrd1 数据的几个示例。该图是使用管道生成的bedgraph文件和python GenomeBrowser软件包（https://pypi.org/project/GenomeBrowser/1.6.3/）准备的，我们旨在简化数据的出版质量基因组浏览器图像的制作。灰色矩形表示表达非编码RNA的基因组区域，例如隐秘不稳定转录本（CUTE），稳定未表征转录本（SUTs）40和Xrn1敏感不稳定转录本（XUT）41。 图6 中的数据表明，Nrd1与许多这些非编码RNA转录本结合，这与该蛋白质参与这类转录本降解的观点一致42。 图6A 显示了IV染色体上的~15 kb区域。在这里，Nrd1与编码高亲和力葡萄糖转运蛋白HXT6和HXT7的转录本的结合显着增加，这两者都在葡萄糖饥饿期间上调。NNS复合物的转录终止可能会影响葡萄糖饥饿期间这些基因的诱导动力学。 图6B 显示了Nrd1与Imd3转录本交联的示例，已知Imd3转录本受Nab343调节。在这种情况下，数据显示葡萄糖饥饿的结合显着减少。先前的工作显示，在葡萄糖饥饿期间，Nab3与Tye7转录本的结合减少44。与这一观察结果一致，χCRAC 数据表明，Nrd1 的结合在葡萄糖饥饿期间降低，Nrd1 与 Tye7 的交联在胁迫 8 分钟后处于最低水平（图 4C）。然而，这种效应似乎只是短暂的，因为在葡萄糖饥饿14分钟后，Nrd1结合回到起始水平。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61027/61027fig1v2.jpg"> 图1: χCRAC 协议的示意图。将标记的菌株生长至所需密度。RBP 表示 RNA 结合蛋白。之后，取参考样品并用254nm紫外光交联。剩余的细胞通过过滤收获，然后迅速转移到应激诱导培养基中。对于此处描述的χCRAC实验，取样并在移位后1，2，4，8，14和20分钟交联（1）。然后使用高度严格的两步亲和纯化纯化目标RBP（2）。接下来，捕获的交联RNA用RNA酶部分消化，在5'端进行放射性标记，并将接头连接到它们上（3）。5'适配器包含独特的"读取中"条形码序列，因此可以在测序后通过生物信息学分离单个样品。然后将RBP-RNA复合物洗脱、汇集并沉淀在一起（4），通过SDS-PAGE分离并通过放射自显影可视化（5）。随后，从凝胶（5）上切下含有主条带正上方放射性信号的单个凝胶切片，在放射自显影图像中用虚线的红色框表示。用蛋白酶K处理凝胶切片，随后提取RNA（6），转化为cDNA，并通过PCR扩增（7）。PCR步骤引入了额外的条形码（P7寡核苷酸引入的黄色块），因此可以将许多文库多路复用到单个泳道中。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61027/61027fig1v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61027/61027fig2v2.jpg"> 图 2:交联和真空过滤。 （A）交联剂。将细胞悬液倒入位于机器右上角的漏斗中（特写另见 图3A ），并保存在位于中间托盘的紫外线透明袋中。该袋子的两侧有两个百叶窗，在用户指示机器开始照射步骤之前，它们一直保持关闭状态。细胞用来自上方和下方托盘的紫外线照射。该机器配有 254 和 365 nm 紫外灯，后者适用于 PAR-CLIP 实验。机器通过位于右上角的触摸屏面板进行操作，该面板允许控制紫外线剂量或曝光时间。（B）交联后，细胞从机器的左侧排出。细胞悬浮液通过真空回收并排入玻璃烧瓶中，随后可以将其倒入真空过滤装置中进行收获。（三）真空过滤装置。它们通过夹子打开和关闭，并在它之间插入一个过滤器。四个过滤装置在非常短的时间序列中并行使用，以免因更换过滤器而损失任何时间。（D）过滤后，将培养基上清液排入烧瓶中进行后续处置。阀门安装在真空过滤装置下方，以在移除过滤器时保持系统中的真空。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61027/61027fig2v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61027/61027fig3v2.jpg"> 图3:交联悬浮细胞与贴壁细胞。 （A）用于悬浮细胞的带有Vari-X接头室的交联剂。将细胞培养物倒入位于托盘右上角的样品入口（漏斗）中。（B）可以容纳塑料或石英培养皿的托盘，用于交联贴壁细胞或少量悬浮细胞。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61027/61027fig3v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61027/61027fig4v2.jpg"> 图 4:文库制备。 （A）来自Nrd1-HTP χCRAC实验的放射自显影示例。强而集中的信号表示与非常短的RNA交联的蛋白质，而上面的涂片表示与足够长度的RNA交联的蛋白质以进行测序。（B）涂片被切除，如凝胶切除后采集的放射自显影所示。（C）来自χCRAC cDNA文库的代表性qPCR。在本例中，cDNA的最大扩增在16个循环中达到。因此，使用16个循环进行最终扩增。误差条表示三个技术qPCR重复的标准偏差。（D）来自6%TBE凝胶上的cDNA文库的磷图像示例。（E）来自芯片的毛细管电泳的cDNA长度和质量分析。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61027/61027fig4v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61027/61027fig5v2.jpg"> 图 5: 高 RNase 测试 iCLIP 实验以测试哺乳动物细胞中的交联。 图中显示了来自GFP-RBM7 iCLIP实验的放射自体图，该实验测试了各种交联能量下RNP回收的效率。使用抗GFP抗体与稳定表达GFP-RBM7的交联细胞上的磁珠偶联进行免疫沉淀。将免疫沉淀物与高浓度的RNase I一起孵育，以便将相关RNA修剪成短而均匀的长度。RNP通过 32P标记和SDS-PAGE可视化，并作为定义的条带迁移，接近非交联蛋白的迁移。定量表明放射性标记的RBM7-RNA信号的密度分析结果归一化为抗GFP蛋白质印迹信号。（A）常用的UVP交联剂与我们的交联剂（Vari-X-linker;（B）我们的交联剂在石英（左）和塑料（右）培养器上的交联时间过程。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61027/61027fig5v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61027/61027fig6v2.jpg"> 图 6:示例基因组浏览器图显示了 χCRAC 显示 Nrd1 与其靶标的差异、时间结合的能力。每个框显示单个基因组区域的图。箭头表示基因在哪条链上编码（左箭头=减链;右箭头=加链）。时间点（分钟）在每个子图的 y 轴上由 t0、t1、t2 等表示。显示了指示染色体和坐标的罗马数字。（A）在葡萄糖剥夺时，Nrd1结合两种高亲和力葡萄糖转运蛋白HXT6和HXT7，它们在这种情况下都上调。（B）观察到Nrd1与Imd3结合，Imd3是Nab344已经验证的靶标，在葡萄糖饥饿后强度降低。（C）Tye7的Nrd1结合表现出动态和短暂的性质，在葡萄糖饥饿后在胁迫8分钟后降至最小。然而，结合随后在14分钟后恢复到基础水平。读取被标准化为"每百万读取数"（RPM;y 轴）。灰色框表示编码非编码RNA的区域。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61027/61027fig6v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using Ï‡CRAC at JoVE.com

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

cDNA的动力学交联和分析是一种允许以高时间分辨率研究活细胞中蛋白质-RNA相互作用动力学的方法。这里详细描述了该方案，包括酵母细胞的生长、UV交联、收获、蛋白质纯化和下一代测序文库的制备步骤。

使用χCRAC以高时间分辨率监测体内蛋白质-RNA相互作用动力学

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

monitoring-protein-rna-interaction-dynamics-vivo-at-high-temporal

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

4.9K 次观看.  University of Edinburgh. cDNA的动力学交联和分析是一种允许以高时间分辨率研究活细胞中蛋白质-RNA相互作用动力学的方法。这里详细描述了该方案，包括酵母细胞的生长、UV交联、收获、蛋白质纯化和下一代测序文库的制备步骤。

视频: Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

科研

生物化学

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

遗传学

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

mRNA从植物原生质体捕获

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

定量细菌中蛋白质-RNA结合的测定

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

生物学

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor

In-cell NMR is a unique approach to observe the structural and dynamic properties of biological macromolecules at atomic resolution directly in living cells. Protein folding, chemical modifications, and conformational changes induced by ligand binding can be observed. Therefore, this method has great potential in the context of drug development. However, the short lifetime of human cells confined in the NMR spectrometer limits the application range of in-cell NMR. To overcome this issue, NMR bioreactors are employed that can greatly improve the cell sample stability over time and, importantly, enable the real-time recording of in-cell NMR spectra. In this way, the evolution of processes such as ligand penetration and binding to the intracellular protein target can be monitored in real time. Bioreactors are often limited by low cell viability at high cell numbers, which results in a trade-off between the overall sensitivity of the experiment and cell viability. We recently reported an NMR bioreactor that maintains a high number of human cells metabolically active for extended periods of time, up to 72 h. This setup was applied to monitor protein-ligand interactions and protein chemical modification. We also introduced a workflow for quantitative analysis of the real-time NMR data, based on multivariate curve resolution. The method provides concentration profiles of the chemical species present in the cells as a function of time, which can be further analyzed to obtain relevant kinetic parameters. Here we provide a detailed description of the NMR bioreactor setup and its application to monitoring protein-ligand interactions in human cells.

This protocol describes the setup of an NMR bioreactor to keep encapsulated human cells viable for up to 72 h, followed by time-resolved in-cell NMR data acquisition and analysis. The methodology is applied to monitor intracellular protein-ligand interactions in real time.

使用高细胞密度生物反应器通过实时定量细胞内 NMR 监测人细胞中蛋白质-配体相互作用

In-cell NMR is a unique approach to observe the structural and dynamic properties of biological macromolecules at atomic resolution directly in living ...

化学

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - 全转录组，蛋白质与RNA相互作用的映射与个别核苷酸分辨率

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...