这项工作是由新加坡NRF奖学金（NRF-2011NRF-NRFF001-025）以及CBRG（NMRC / CBRG / 0070/2014）的资助。

注:在本研究的上下文中感兴趣的RNA为AR 3&#39;UTR的片段。 1.生物素标记的RNA的制备<ol><li>以获得的RNA，使用商业试剂盒和以下生产商的说明11通过PCR扩增T7-AR-低聚，随后通过体外转录。 注:引物用于PCR列于表4:T7-AR-F和T7-AR-R。 </li><li>对于非特异性结合控制，通过PCR扩增的萤火虫萤光素酶（FL）的DNA psiCHECK-2的质粒，随后通过体外转录的部分序列。 注:引物用于PCR列于表4:hluc（+） - T7探针-F和hluc（+） -探针- R 表4模板和引物进行PCR扩增的序列。 </li><li>对于50微升的PCR反应，混合50纳克DNA（寡核苷酸或质粒），4微升的dNTP（每个人的dNTP为2.5毫米），5微升10X反应缓冲液，1微升2.5 U /＆＃181;微升DNA聚合酶，2微升10μM的正向引物，2微升10μM反向引物，和无核酸酶水。 <ol><li>使用下列程序运行在热循环仪进行PCR扩增。第1步:95℃2分钟一个循环;第2步:35个循环的95℃30秒，55℃30秒和72℃30秒（AR）或60秒（佛罗里达州）; STEP3:10分钟，72℃的一个周期。 </li></ol></li><li>合成使用以下制造商的说明11 的体外转录试剂盒的生物素化的RNA。在该实验中，使用PCR产物作为用于RNA合成的模板。对于一个20微升的体外转录反应，混合200纳克的PCR扩增的DNA，2微升每个单独的NTP的，1微升10mM的生物素的CTP，2微升10×反应缓冲液，和2μlT7 RNA聚合酶。孵育在37℃下该混合物4小时。 </li><li>在37＃以下体外转录，用1微升2单位/微升DNA酶治疗反应混合物176℃持续15分钟，以降解模板DNA。最后，稀释生物素化的RNA，以60微升为随后的纯化的总体积。 </li><li>纯化生物素化的RNA，通过使用以下制造商的说明12纯化柱以除去盐和未掺入的核苷酸。测量RNA浓度，并储存于-80℃用于后续的下拉测定。 </li><li>至在90℃下确保在RNA-蛋白质相互作用，热加入50μl生物素化的RNA（50时）感兴趣的RNA适当的二级结构2分钟，冰浴2分钟，然后用50μl2×RNA结构缓冲液（ 表提供3），并允许将RNA在RT折叠30分钟13,14。 </li></ol> 2. RNA结合的珠的制备注:使用磁立场的磁珠和上清液分离。 <ol><li>通过简短vorte悬浮在原始小瓶链菌素包被的磁珠兴下列制造商的说明。转移150微升重新悬浮珠子到1.5ml管中。放置在磁体的管1分钟。弃上清。保持珠沉淀。 注:在RNA Pull-down实验，由原来的小瓶等分悬浮颗粒时，使用的检测之后印迹50微升珠和200微升珠化验后MS。 </li><li>通过重新悬浮珠粒料用1ml制造商推荐的1×W＆的B（结合和洗涤）缓冲液（ 表3）洗一次珠，随后在旋转器在室温下旋转管5分钟。弃上清。保持珠沉淀。 </li><li>通过重新悬浮珠粒料用400μl缓冲液A缓冲液A灭活珠上的RNase，洗珠两次，每次是含有0.1M NaOH和0.05 M氯化钠（ 表3）的碱性溶液。旋转管，在室温下在旋转器2分钟。弃上清。保持珠沉淀。 </li> &lt;li&gt;要通过重新悬浮珠粒料用400μl缓冲液B（ 表3），接着在一旋转器在室温下旋转管2分钟从珠除去氢氧化钠，洗珠两次，每次的时间。弃上清。保持珠沉淀。 </li><li>用300微升2倍W＆B缓冲液重悬珠颗粒，分割珠平等，转移成三个1.5毫升管（每管100微升珠）。供应含珠管100微升核糖核酸酶自由水，100微升生物素化FL RNA和100​​微升生物素化AR 3&#39;UTR RNA，分别为。 </li><li>孵育各胎圈混合物在室温下用旋转1小时。放置管上的磁铁1分钟。丢弃所有上清。保持珠颗粒。 </li><li>通过重新悬浮用400μl1×W＆B缓冲液各胎圈粒料洗涤珠两次，每次，随后在一旋转器在室温下旋转管5分钟。 </li><li>放置管上的磁铁1分钟。丢弃所有上清。重悬每个珠子PELLE吨，50微升核隔离缓冲液（ 表3）。 </li></ol> 3.前脂肪细胞的分离和脂肪生成诱导<ol><li>正如在以前的出版物5描述，剖析从肩胛间棕色脂肪垫前脂肪细胞（C57BL6野生型小鼠，3周龄）。 </li><li>生长并在体外分化前脂肪细胞5。细胞准备用于分化开始后4-5天的下游应用。 </li></ol>从初级脂肪细胞的细胞裂解物的制备4 注:确保细胞裂解液的制备所有的程序都在冰制成，和所有缓冲区，在冰上冷却。冰上预寒意DOUNCE玻璃均质。 <ol><li>成长并在15 cm的培养皿5（参见3.2节）区分脂肪前体细胞。每道菜都提供了足够的细胞一个RNA pull-down实验。在第4天或分化，丢弃细胞培养基的第5天，和洗涤策LLS一旦与1X PBS。 </li><li>收获细胞，加入10-15毫升1×PBS中至细胞，刮去离心200 XG收集在50ml管中的细胞，并沉淀细胞在RT 5分钟。用1毫升吸管弃上清。 </li><li>在2毫升悬浮细胞沉淀低渗缓冲液（ 表3），并在冰上孵育细胞15分钟。转移细胞到15毫升DOUNCE玻璃匀浆和剪切细胞机械地在冰上用20笔。 </li><li>沉淀细胞核离心3300 xg离心15分钟，在4℃。保持冰为进一步利用细胞核沉淀（参见4.6节）。转移上清液至1.5毫升管中，添加3M氯化钾到上清液以20,000 xg离心以获得150mM的终浓度，并旋转，在4℃下15分钟。 </li><li>离心后，小心地用1毫升吸管除去lipidlayeron顶部，并收集上清液作为细胞质细胞裂解物。 </li><li>重悬细胞核沉淀（见4.4）在1毫升核隔离缓冲。 ŧ转让（BOT）的细胞核用1毫升吸管15毫升DOUNCE玻璃匀浆，并机械上用100笔划冰剪切核。沉淀通过离心核碎片以20,000 xg离心15分钟，在4℃，并收集上清液作为核细胞裂解物。 </li><li>无论是结合了核和细胞质的细胞裂解物，或单独使用每个分数，下游下拉应用程序。在此演示实验中，细胞裂解物的这两个级分合并以1:1的体积比为1:1。 </li><li> RNase抑制剂添加到该未分级的细胞裂解物，以获得0.5U /微升的最终浓度。抛开100微升细胞裂解液作为免疫印迹15输入控制。 </li></ol> 5.绑定和RBP洗脱<ol><li>分裂细胞裂解物分成三个等份（在1.5ml管1毫升细胞裂解物），并与空白孵育，佛罗里达的RNA共轭和AR 3&#39;UTR的RNA共轭珠粒（见2.8节），分别为3小时，在4℃ C轮换。 </li><li>放置管上的磁铁1分钟。使用1毫升吸管，随后RNA分离从上清液来确认RNA的完整性收集上清液。通过使用以下制造商的说明（ 表2）的酸性硫氰酸胍-苯酚溶液分离RNA。通过分析核糖体RNA的28S和18S亚基评估RNA的完整性。保持冰珠颗粒。 </li><li>通过重新悬浮每个珠子粒料用含有40单位RNA酶抑制剂和0.25％IGEPAL，随后在旋转器在室温下旋转管2分钟1毫升核隔离缓冲器洗涤磁珠六次，每次。对于使用的磁珠和上清液分离磁立场。丢弃所有上清。保持冰珠颗粒。 </li><li>使用以下步骤，从免疫印迹珠洗脱RNA-蛋白复合物。首先，在悬浮75微升核隔离缓冲每个珠子颗粒;然后，加入25微升4倍免疫印迹装载BUFFER（含有SDS），得到100微升的总体积。最后，在95℃下煮沸此100μl的溶液珠5分钟。 注意:使用以下步骤，从MS珠洗脱RNA-蛋白复合物。第1步:重悬在100μl洗脱缓冲液（ 表3）每个珠子粒料;第2步:孵育珠样品在室温下用旋转2-3小时; STEP3:离心机，并收集含蛋白质的上清液; STEP4:提交的MS之前集中蛋白质溶液20-30微升。 </li><li>离心机每100微升珠样品以12,000×g离心30秒，在4℃，并且收集上清液。分装15微升上清免疫印迹分析（见6.1和6.2）。探测用稀释的HuR小鼠单克隆抗体的得到的膜（1:1,000稀释度），过夜。 </li></ol> 6.免疫印迹的RBP验证<ol><li>独立的限制性商业惯例，通过SDS-PAGE使用标准技术。 </li><li>我们开展通过探测与感兴趣的RNA相关的限制性商业惯例用标准技术船尾印迹。 注:在这个演示实验中，使用了的HuR蛋白的免疫检测的HuR鼠单克隆抗体。 </li><li>孵育稀释的HuR抗体所得膜:在5％（1 1000稀释）w / v的脱脂奶粉，1×TBS， 0.1％ 吐温- 20日4 °下缓慢摇动过夜。用1×TBST洗膜三次。在室温下1小时的第二抗体（山羊抗 - 小鼠IgG-HRP）孵育膜。洗膜。制定并记录信号。 </li></ol>

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lncRNAs和限制性商业惯例在健康和疾病的重要角色，但这些分子的分子机制知之甚少。与lncRNA分子相互作用的蛋白的鉴定是朝着阐明的调节机制的关键步骤。通过将RNA下拉测定系统限制性商业惯例的富集是基于在溶液中相互作用的复杂，使得从细胞裂解物蛋白可使用结合于链霉抗磁珠的生物素化的RNA诱饵被选择性地提取的捕获。其他一些方法，如啁啾，图表，RAP可以达到同样的目的，但这些方法?...

A correction to the author list was made to: <a href="http://www.jove.com/video/54207/detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte">Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture</a>.
The author list has been updated from:
Qianfan Bai1*, Zhiqiang Bai1*, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
*These authors contributed equally
to:
Qianfan Bai1*, Zhiqiang Bai1*, Shaohai Xu3, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
3Division of Bioengineering, School of Chemical &#38; Biomedical Engineering, Nanyang Technological University
*These authors contributed equally

A correction to the author list was made to: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture.

Erratum: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

限制性商业惯例是结合在细胞中的双链或单链RNA和参与形成RNA-蛋白复合物的蛋白质。限制性商业惯例可以结合多种RNA分子，包括mRNA和长非编码RNA（lncRNAs），并在发挥转录后水平的影响力。限制性商业惯例和lncRNAs在脂肪发展逐渐成为新的监管机构和功能1,2,3。理解RBP-的机制和细胞途径lncRNA介导的调节，它通常需要检测特定RNA分子和一个或多个限制性商业惯例之间的相互作用，并且，有时，以识别RNA的蛋白伴侣的全谱成绩单。然而，该实验可以是具有挑战性由于脂质的脂肪细胞的高含量。这里所描述的RNA的下拉协议可被用于检索来自原代培养4,5的脂肪细胞裂解物的特定RNA的蛋白质的合作伙伴。 理这个原山口总结如下。的RNA诱饵是在体外从DNA模板转录，生物素标记和缀合到链霉抗包被的磁珠4。所述RNA诱饵与细胞裂解物孵育以允许RNA-蛋白质复合物，其随后被拉下来的磁性支架的形成。更具体地，该RNA下拉协议如下所述使用的RNA片段从AR 3&#39;UTR作为诱饵检索的HuR蛋白质，普遍表达RBP势必的mRNA 6,7的3&#39;UTR，从theadipocytelysate原代培养。为了测试该测定的结合特异性，非相关RNAas以及空白链霉珠被包括作为对照。此协议是与蛋白质印迹或质谱（MS）兼容，以确认一个特定RBP的捕获或识别捕获的限制性商业惯例，分别为8的全剧目。 几种技术可用于研究RNA-蛋白质相互作用秒。揭示通过一个给定的RBP，RIP（RNA免疫沉淀）和CLIP（UV交联和免疫沉淀）结合的RNA可以应用。与此相反，以确定一个给定RNA的蛋白质伙伴，RNA下拉，啁啾（通过RNA纯化染色质分离），图表（RNA靶捕获杂交分析）和RAP（RNA反义纯化）都可以应用。在与后来者相比，RNA下拉技术花费较少的精力来设置。它可以用来捕获蛋白在体外 和体内 。 RNA的下拉，这在技术上是比其体外对应更具挑战性， 在体内的方法，通过在细胞中的交联保留RNA-蛋白质相互作用，捕获的从细胞中感兴趣的适体-标记的RNA，和随后检测结合的限制性商业惯例。 RNA的下拉可以用于充实低丰度限制性商业惯例，并且分离和鉴定在控制细胞调节9,10具有多样功能作用的RNA-蛋白质复合</s了&gt;。

<table><tbody><tr><td>&lt;strong&gt;Major materials used in this study.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MEGAscript kit</td><td>&amp;nbsp;Ambion</td><td></td><td></td></tr><tr><td>Biotin-14-CTP&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>NucAway spin columns&amp;nbsp;</td><td>Ambion</td><td></td><td></td></tr><tr><td>Dynabeads M-280 Streptavidin&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>HuR (3A2) mouse monoclonal antibody (sc-5261)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td></td><td></td></tr><tr><td>Goat anti-mouse IgG-HRP(sc-2005)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td></td><td></td></tr><tr><td>Hypure Molecular Biology Grade Water (nuclease-free)&amp;nbsp;</td><td>HyClone</td><td></td><td></td></tr><tr><td>Phosphate Buffer Saline (1&amp;times;PBS)</td><td>&amp;nbsp;HyClone</td><td></td><td></td></tr><tr><td>RNase inhibitor&amp;nbsp;</td><td>Bioline</td><td></td><td></td></tr><tr><td>Protease inhibitor&amp;nbsp;</td><td>Sigma</td><td></td><td></td></tr><tr><td>Pfu Turbo DNA polymerase&amp;nbsp;</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>TRIsure</td><td>&amp;nbsp;Bioline</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Major equipment used in this study.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sorvall Legend Micro 21R centrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Sorvall Legend X1R centrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Bio-Rad T100 thermal cycler&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Nanodrop 2000&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>BOECO rotator Multi Bio RS-24&amp;nbsp;</td><td>BOECO,Germany</td><td></td><td></td></tr><tr><td>Protein gel electrophoresis and blotting apparatus&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>DynaMag-2 magnet&amp;nbsp;</td><td>Life Technologies</td><td></td><td></td></tr></tbody></table>

detection of rna-binding proteins by in vitro rna pull-down in adipocyte culture

RNA结合蛋白（限制性商业惯例）正在成为在脂肪的发育和功能的调节层。限制性商业惯例通过影响靶mRNA的稳定性和翻译效率发挥在转录后水平的基因表达的调控中起关键作用。 RNA的下拉技术已被广泛用于研究RNA-蛋白质相互作用，这是必要阐明该机制底层限制性商业惯例&#39;以及长的非编码RNA"（lncRNAs）函数。然而，高血脂丰度在脂肪细胞构成在进行该实验中一个技术挑战。这里详细的RNA下拉协议是初级脂肪细胞培养进行了优化。从雄激素受体的（AR）3&#39;非翻译区含作为一个例子来说明如何检索其RBP合作伙伴的HuR蛋白质，脂肪细胞从一个lystate富含腺苷酸尿苷酸-elementwas（3&#39;UTR）的RNA片段。可以应用这里描述的方法来检测之间的相互作用限制性商业惯例和非编码RNA，以及间限制性商业惯例和编码RNA。

在这个演示实验中，AR RNA片段作为诱饵捕捉到它的结合蛋白的HuR。两者FL的RNA诱饵即非相关的HuR蛋白，以及未缀合的链霉空白珠的等分试样用作阴性对照。 RNA-蛋白的相互作用，可能会发生无论是在细胞核或细胞质中，并且此下拉协议可以应用到无论是总或分级（核或细胞质）细胞裂解物。通过Western印迹的蛋白质的分析表明，15微升未分级细胞裂解物的样品（见4.8节），如...

Watch this Scientific Journal Video about Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture at JoVE.com

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

的RNA下拉协议这里优化用于检测RNA结合蛋白（限制性商业惯例）和非编码以及编码RNA之间的相互作用。从雄激素受体（AR）的RNA片段作为例子来演示如何从初级的棕色脂肪细胞的lystate检索其RBP。

RNA结合蛋白的检测通过<em&gt;体外</em在脂肪细胞培养&gt; RNA下拉

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte

Research

JoVE Journal

Biology

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

23.5K 次观看.  Duke-NUS Medical School. 的RNA下拉协议这里优化用于检测RNA结合蛋白（限制性商业惯例）和非编码以及编码RNA之间的相互作用。从雄激素受体（AR）的RNA片段作为例子来演示如何从初级的棕色脂肪细胞的lystate检索其RBP。 

视频: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

科研

遗传学

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

生物化学

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 ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

rna 下拉法识别长非编码 rna 的 rna 靶点

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Desthiobiotin labeling of a synthetic 25-nucleotide RNA oligo, which contains an adenine-rich element (ARE) motif, allows specific binding of cytosolic ARE-binding protein.

Desthiobiotin-链亲和素亲和介导纯化间皮瘤细胞 RNA 相互作用蛋白的研究

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

癌症研究

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

基于生物素的下拉法对细胞 miRNAs mRNA 靶点的验证

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

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 ...

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

使用短生物素化RNA对RNA结合蛋白进行优化的定量下拉分析

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

研究蛋白质和微RNA调制对脂肪细胞功能的影响的脂肪细胞培养模型

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

RNA结合蛋白的检测通过

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