Name: electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example
Uploaded: 2014-12-03T16:00:00.000+00:00
Duration: 12 min 44 s
Description: Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

小鼠实验程序批准了麦吉尔大学（协议4966）的动物护理委员会。 1.准备从培养细胞蛋白提取物<ol><li>用10毫升冰冷的磷酸盐缓冲盐水（PBS）洗两次培养的细胞。 </li><li>刮贴壁细胞与任一橡皮或塑料细胞刮棒在1ml冰冷的PBS，转印悬浮到1.5ml微量离心管中。 </li><li>旋在微量离心5分钟，于700×g离心，于4℃。吸PBS。 </li><li>加入100微升冰冷的细胞质裂解缓冲液（ 见表1）每10 7个细胞，并吸取向上和向下。 </li><li>在冰上孵育20分钟。 </li><li>旋，在4℃下以全速10分钟在微量。 </li><li>丢弃沉淀。上清转移到新的1.5 ml离心管，并保持在冰上。 </li><li> DET使用Bradford法23 - （10微克/微升通常1）貂皮蛋白浓度。 </li><li>等分试样并储存细胞提取物在-80℃下直到使用。 </li></ol> 2.准备从小鼠肝脏和脾脏蛋白提取物<ol><li>安乐死鼠标使用CO 2吸入。 </li><li>奠定安乐死的动物在一块干净的纱布在解剖板。打开用剪刀腹部。 </li><li>解剖肝脏和脾脏用剪刀和镊子，并冲洗在大约50毫升的冰冷的PBS各组织。 </li><li>立即切割组织切成小块，用手术刀（例如:约1 - 2 立方毫米）。 </li><li>无延迟，放在新鲜冻存管组织的片，然后捕捉冻结他们在液氮。商店管理单元冷冻组织等分在-80℃下直到使用。 </li><li>在0.25 - （2 mm 3的大约1） - 0.5毫升匀化一片冷冻组织的冰冷细胞质裂解缓冲液（ 见表1）用组织匀浆10秒。 </li><li>转移匀浆至1.5 ml离心管，并冰浴20分钟。 </li><li>旋，在4℃下以全速10分钟在微量。 </li><li>放弃颗粒和转移上清到新的1.5 ml离心管。保持在冰上。 </li><li>测定蛋白浓度-使用Bradford测定法23（通常为1 10微克/微升）。 </li><li>等分试样并储存细胞提取物在-80℃下直到使用。 </li></ol> 3.准备放射性标记IRE-探头<ol><li>通过在37℃下用限制性核酸内切酶XbaⅠ位（每质粒微克1 U）的IRE序列的下游其裂解温育1小时进行线性化的含有质粒IRE I-12.CAT 22。将线性化质粒将被用作模板的体外转录。 </li><li>设置一个中的 n 体外在20微升的总体积的转录反应。使用表2中所示的储液，并添加1微升线性质粒模板，4微升的转录缓冲液1微升混合物中ATP / CTP / GTP混合，加入10μl[α-32 p] -UTP，2微升二硫苏糖醇，1微升RNA酶抑制剂和1μlT7 RNA聚合酶。通过上下吹打混匀。 </li><li>孵育在40℃下1小时24。 </li></ol> 4.净化放射性标记IRE-探头<ol><li>加入1微升的0.5M EDTA，pH为8混音通过上下抽吸终止的体外转录反应。 </li><li>添加10微升10毫克/毫升的tRNA，作为载体更好沉淀。通过上下吹打混匀。 </li><li>添加82.5微升的3M乙酸铵。涡旋混合。 </li><li>添加273微升乙醇。涡旋混合。 </li><li>静置在室温5分钟。 </li><li>在微型在RT旋转10分钟以全速。弃去上清液。 </li><li>洗涤沉淀用100μl的70％乙醇。 </li><li>在微型在RT旋转10分钟以全速。弃去上清液。 </li><li>空气干燥沉淀10分钟。 </li><li>在100μl双蒸重悬沉淀，预先高压灭菌的 H 2 O. </li><li>量化放射性用液体闪烁计数器，等分试样的放射性标记的探针的IRE并储存在-80℃直到使用。冷冻等分试样可用于长达3周。 </li></ol> 5.准备EMSA原生聚丙烯酰胺凝胶<ol><li>通过使用1.5毫米垫片和梳组装凝胶（16×16厘米）。 </li><li>以制备6％非变性聚丙烯酰胺凝胶，使用表3中所示的储备溶液混合7.5毫升40％的丙烯酰胺:双丙烯酰胺，加入5ml 5×TBE和37.5毫升双蒸H 2 O. </li><li>加入0.5毫升10％的新制备的过硫酸铵（APS）和25微升四甲基乙二胺（TEMED）。 </li><li>立即倒入丙烯酰胺溶液的凝胶，并让它聚合。等待大约30分钟。 </li><li>与凝胶组装电泳装置中，填充罐用0.5X TBE并连接电源。 </li></ol> 6.凝胶电泳迁移分析<ol><li>在10微升的总体积稀释25微克从细胞或组织与细胞质裂解缓冲液（ 表1）的蛋白提取物（低蛋白质浓度也可以使用）。保持在冰上。如果需要的话，加入1微升1:4稀释的2-巯基乙醇（2-ME），以激活休眠IRP1（终浓度:2％）25。 </li><li>稀释放射性IRE探头双蒸H 2 O来200000 CPM /μL，热在95℃变性℃1分钟，并冷却在RT至少5分钟。 </li><li>成立EMSA反应通过加入1微升放射性IRE探针与蛋白质提取物。 </li><li>孵育20分钟，在RT。 </li><li>加入1微升50毫克/毫升肝素的反应（以抑制非特异性蛋白相互作用与探针9），并继续孵育另外10分钟。分装肝素在-80℃下储备溶液（50毫克/毫升），并存储。 </li><li>当使用放射性标记的长探针（&gt; 60个核苷酸），加入1微升RNA酶T1（1U /微升），孵育10分钟，在室温，以减少非特异性蛋白结合到探针，并允许更好地分离的RNA /蛋白质的电泳过程中的复杂。 </li><li>加入3微升加样缓冲液（80％甘油+溴酚蓝），混合和负载上的6％非变性聚丙烯酰胺凝胶。 </li><li>运行凝胶在130 V（5伏/厘米）60分钟。 </li><li>牛逼转输的凝胶到大滤纸和干燥。 </li><li>暴露于膜和开发放射自显影。暴露时间的范围可以从1小时（或更少）至O / N。 </li></ol>

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The authors declare that they have no competing financial interests.

在此，我们描述了已开发研究IRP1和IRP2的IRE结合活动的协议，并且我们显示代表性数据。通过使用不同的探针，该协议也可以被用于其它RNA结合蛋白的研究调整。一个关键步骤是探针的大小。用法长探针，这是共同的时，确切的结合位点是未知的，可能会导致的RNA /蛋白质复合物不不同迁移比游离的RNA。在这种情况下，最好是通过用RNA酶T1（ 步骤6.6）处理以去除未结合的RNA。探针的质?...

在EMSA最初被开发，研究DNA结合蛋白与靶DNA序列1,2的关联。其原理是类似的RNA /蛋白质相互作用3，这是本文的重点。简言之，RNA被带负电荷并朝向阳极期间非变性电泳聚丙烯酰胺（或琼脂糖）凝胶将迁移。凝胶内的迁移取决于核糖核酸，其正比于它的电荷的大小。相比于自由RNA特异性的蛋白质的RNA的结合改变其流动性，且复杂的迁移变慢。这主要是由于增加的分子量，而且要在电荷和可能的构象的改变。利用RNA标记为探针，可以方便地监控了"凝胶阻滞"或"条带迁移"的。的32 P标记的RNA探针的使用是很常见的，并提供高灵敏度。被检测的RNA /蛋白质复合物和游离RNA的迁移通过放射自显影。缺点是:32 P（14.29天），探针的质量由于辐逐渐恶化的半衰期短，一个放射性许可证和基础设施为放射性工作，以及潜在的生物安全的担忧的要求。因此，已经开发了替代非同位素方法用于标记的RNA探针，例如用荧光团或生物素，其能够检测由荧光或化学发光成像4,5。这些方法的局限性是成本较高并且经常敏感性降低相比同位素标记和非同位素标记物的潜在干扰的RNA /蛋白质相互作用。非变性聚丙烯酰胺凝胶适用于大多数应用EMSA和常用。有时，琼脂糖凝胶可能造成的大型复合物的分析，另一种选择。 EMSA的主要优点在于，它结合了简单，灵敏度和鲁棒性4 &lt;/ SUP&gt;。该测定可以在几个小时内完成，并且不需要复杂的仪器。 RNA /蛋白质相互作用可通过EMSA在浓度低至0.1纳米或更小进行检测，并广泛的结合条件内（pH为4.0 - 9.5，单价盐浓度1 - 300毫米，而温度0 - 60℃）。 RNA /蛋白质复合物的形成，也可以由滤波器结合测定研究。这是基于对RNA /蛋白质复合物中的硝化纤维过滤器中的保留一个简单，快速，和便宜的程序，而自由RNA探针穿过6。相比EMSA，它的情况下将RNA探针含有多个结合位点，或粗提取物包含一个以上的RNA结合蛋白结合的探针在同一地点的限制。而多的RNA /蛋白质相互作用会逃过检测由滤波器结合测定中，它们可以容易地通过EMSA观察。在某些情况下，可视化前夕n个可能时两个RNA /蛋白质复合物共迁移（例如，人类IRP1 / IRE和IRP2 / IRE复合物）中，加入的抗体对RNA结合蛋白的EMSA反应之一，在凝胶上产生进一步的延迟（ "超迁移"）7。 在EMSA已广泛用于研究IRP1和IRP2，这是铁代谢8-10转录后调节器。它们的工作由几个11的mRNA的非编码区中结合IRES，系统发育保守的发夹结构。 IRE有时首先在编码铁蛋白12和转铁蛋白受体1（TFR1）13，铁贮存和吸收，分别为蛋白质的mRNA的发现。稍后，IRE有时被发现在红细胞特异性氨基乙酰丙酸合成酶 （ALAS2）14，线粒体乌头15，铁转运蛋白16， 二价金属转运蛋白1（DMT1）17，缺氧诱导因子2 <e米&gt;α（HIF2α）18，以及其他的mRNA 19-21。原型H和L-铁的mRNA包含在其5 1 IRE&#39;UTR，而TFR1基因包含在其3多IRE有时&#39;UTR。 IRE / IRP的相互作用特异性抑制铁蛋白mRNA的翻译在空间上阻断43S核糖体亚基及其相关;此外，他们稳定TFR1反对内切裂解的mRNA。 IRP1和IRP2份额广泛的序列相似性，并表现出铁饥饿细胞高IRE结合活性。在铁充足的细胞，IRP1组装转换它到胞质乌头在其IRE结合活性为代价一立方烷的Fe-S簇，而IRP2经历蛋白酶体降解。因此，IRE / IRP相互作用取决于蜂窝铁状态，但也受其它信号，如H 2 O 2，一氧化氮（NO）或缺氧。在这里，我们描述了协议，以评估IRE-结合活性从原油细胞和组织的提取物用EMSA。我们使用的是通过体外转录从质粒DNA模板（I-12.CAT），其中IRE序列由最初引入有义方向的T7 RNA聚合酶位点下游生成一个32 P-标记的H-铁蛋白的IRE探针克隆退火合成寡核苷酸22。

<table><tbody><tr><td>leupeptin</td><td>SIGMA</td><td>L2884</td><td></td></tr><tr><td>PMSF</td><td>SIGMA</td><td>78830</td><td></td></tr><tr><td>BioRad Protein Assay</td><td>BIORAD</td><td>500-0006</td><td></td></tr><tr><td>T7 RNA polymerase</td><td>Thermoscientific</td><td>EPO111</td><td></td></tr><tr><td>RNase Inhibitor</td><td>Invitrogen</td><td>15518-012</td><td></td></tr><tr><td>UTP [alpha-&lt;sup&gt;32&lt;/sup&gt;P]</td><td>Perkin-Elmer</td><td>NEG507H</td><td></td></tr><tr><td>Scintillation liquid</td><td>Beckman Coulter</td><td>141349</td><td></td></tr><tr><td>heparin</td><td>SIGMA</td><td>H0777</td><td></td></tr><tr><td>Rnase T1</td><td>Thermoscientific</td><td>EN0541</td><td></td></tr><tr><td>Name of the Equipment</td><td></td><td></td><td></td></tr><tr><td>Tissue Ruptor</td><td>Qiagen</td><td>9001271</td><td></td></tr><tr><td>Scintillation counter</td><td>Beckman Coulter</td><td>LS6500</td><td></td></tr><tr><td>Protean II xi Cell</td><td>BIORAD</td><td>165-1834</td><td></td></tr><tr><td>20 wells combs</td><td>BIORAD</td><td>165-1868</td><td>1.5 mm thick</td></tr><tr><td>1.5 mm spacers</td><td>BIORAD</td><td>165-1849</td><td></td></tr><tr><td>PowerPac</td><td>BIORAD</td><td>164-5070</td><td></td></tr></tbody></table>

electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

RNA /蛋白质相互作用是转录后调控途径的关键。间的最佳表征的胞质RNA结合蛋白是铁调节蛋白 ，IRP1和IRP2。它们结合到铁几个靶mRNA的非翻译区（UTR）内响应元件（IRES），从而控制的mRNA翻译或稳定性。 IRE / IRP的相互作用已被广泛研究了EMSA。在这里，我们描述了EMSA协议用于分析IRP1和IRP2的IRE结合活性，这可以推广到其他评估RNA结合蛋白的活性。含有一种RNA结合蛋白，或纯化此蛋白质的制备的粗蛋白裂解物，孵育用过量的32 P标记的RNA探针，从而允许形成复合物。肝素加入到排除非特异性蛋白探查结合。随后，将混合物通过在聚丙烯酰胺凝胶非变性电泳分析。免费探头迁移速度快，而所述RNA /蛋白质复合物表现出延迟的流动性;因此，该过程也被称为"凝胶阻滞"或"条带迁移"测定法。电泳结束后，将凝胶干燥和RNA /蛋白质复合物，以及游离的探针，通过放射自显影检测。该协议的总体目标是检测和量化的IRE / IRP和其它RNA /蛋白质相互作用。此外，EMSA也可用于确定特异性所研究的RNA /蛋白质相互作用，结合亲和力和化学计量。

放射标记的IRE探针制备，如在第3和第4的协议的说明。所述探针的序列为5&#39;-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUGÇUUCAAÇAGUGC UUGGA CGGAUCCU-3&#39;;粗体的核苷酸代表一个未配对的C残基和环，这是至关重要的IRE特征。探针的比放射性为4.5×10 9的cpm / RNA微克。 评估铁扰动对IRE结合活性的影响，鼠的RAW264.7巨噬细胞不进行治疗，或者与血红素（铁源）或去铁胺（铁螯合剂）...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

在这里，我们提出了一个协议分析RNA /蛋白质相互作用。电泳迁移实验（EMSA）是基于RNA的/蛋白复合物和游离RNA在天然凝胶电泳迁移差异上。通过使用放射性标记的RNA探针，RNA /蛋白质复合物可以通过放射自显影进行显现。

凝胶电泳迁移分析（EMSA）为RNA，蛋白质相互作用的研究:IRE / IRP示例

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

53.6K 次观看.  Jewish General Hospital. 在这里，我们提出了一个协议分析RNA /蛋白质相互作用。电泳迁移实验（EMSA）是基于RNA的/蛋白复合物和游离RNA在天然凝胶电泳迁移差异上。通过使用放射性标记的RNA探针，RNA /蛋白质复合物可以通过放射自显影进行显现。 

视频: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

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

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

生物化学

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

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

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 ...

Electrophoretic Mobility Shift Assay for Detection of Transcription Factor-DNA Complexes

Source: Hsieh, Y. W. et al., <a href="https://app.jove.com/v/54863">An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the electrophoretic mobility shift assay to detect protein-DNA interactions using infrared fluorescent-labeled DNA probes. When the protein binds to the DNA probe, it forms a large complex that migrates slowly on and appears as a shifted band on the gel.

用于检测转录因子-DNA 复合物的电泳迁移率变化测定

Source: Hsieh, Y. W. et al., An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides. J. ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-nucleic acid interactions

SELEX-Based In Vitro Binding Assay to Identify RNA-Protein Interactions

Source: Singh, R. <a href="https://app.jove.com/v/59635">Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins.</a> J. Vis. Exp. ( (2019).
In this video, we describe the systematic evolution of ligands by the exponential enrichment (SELEX) method to identify specific RNA-binding sequences for a target protein of interest. The protein is incubated with a large pool of randomized RNA sequences, and the protein-binding RNA sequences are isolated, PCR-amplified, and sequenced to identify their protein-binding sites.

基于 SELEX 的体外结合测定法，用于鉴定 RNA-蛋白质相互作用

Source: Singh, R. Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins. J. Vis. Exp. ( (2019).
In this video, we ...

Mobility Shift Affinity Capillary Electrophoresis: A Method to Analyze Sample-Ligand Interactions Depending on Differential Migration of Protein-Ligand Complexes

Source: Nachbar M. et al., <a href="https://www.jove.com/v/57749">Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis</a>. J. Vis. Exp. (2018).
This video describes the method to analyze protein-ligand interaction by mobility-shift affinity capillary electrophoresis. This method can be used for characterizing the binding behavior of a protein with various charged ligands.

迁移率变化亲和毛细管电泳:一种根据蛋白质-配体复合物的差异迁移分析样品-配体相互作用的方法

Source: Nachbar M. et al., Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary ...