Name: exploring sequence space to identify binding sites for regulatory rna-binding proteins
Uploaded: 2019-08-09T14:00:00
Description: Watch this Scientific Journal Video about Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins at JoVE.com

作者感谢国家卫生研究院过去的资助。

注:图1概述了迭代选择-放大(SELEX)过程中的关键步骤。
1. 生成随机库模板
<ol><li>合成前引物5'- GTAATACGACTACTATAGGGTGATCAATATCCA-3'和反向引物5'- GCGACGGATCCAGCTT CA-3',通过在DNA合成器上的化学合成。 注:引信和随机库可以进行商业合成。</li>
	<li>合成随机库寡核苷酸模板 5'- GGTAtATATCTAtCTCTCTCTCTCTCTCTCCA(N1...N31)通过化学合成,TGAAGCTA...

<ol>
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核酸结合蛋白是动植物发育的重要调节剂。SELEX 程序的一个关键要求是开发可用于分离蛋白质结合和未结合的 RNA 馏分的测定。原则上,此测定可以是体外结合测定,如滤芯结合测定、凝胶移动转移测定或重组蛋白、纯化蛋白或蛋白质复合物的基质结合测定19。测定也可以是酶测定,其中前体和产品(或中间体)可以根据大小或其他某种方式分离20。
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在细胞生物学中,基因调控起着核心作用。在基因表达途径的一个或多个步骤中,基因具有被调节的潜力。这些步骤包括转录(启动、伸长和终止)以及拼接、多化或3'端形成、RNA导出、mRNA翻译和初级转录的衰变/定位。在这些步骤中,核酸结合蛋白调节基因调控。识别这种蛋白质的结合位点是研究基因控制的一个重要方面。突变分析和遗传序列比较已用于发现核酸中的调控序列或蛋白质结合位点,如启动子、拼接位点、聚化元素和转化信号1。2,3,4.
前mRNA拼接是基因表达和调控过程中不可或缺的一步。大多数哺乳动物的基因,包括人类的基因,都有内源基因。这些转录本的很大一部分是交替拼接的,从同一基因或主转录本产生多个mRNA和蛋白质异构体。这些等体在细胞生物学中具有细胞特异性和发育作用。5' 拼接位点、分支点和聚苯乙烯-3'拼接位点是受监管的关键拼接信号。在负调节中,否则强接头位被抑制,而在正调节中,否则弱拼接位点被激活。这些事件的组合会产生大量功能上不同的异形。RNA结合蛋白在这些替代拼接事件中起关键作用。
许多蛋白质是已知的,其结合位点或RNA靶点仍有待确定5,6。将调节蛋白与其下游生物靶点或序列联系起来通常是一个复杂的过程。对于此类蛋白质,识别其靶向RNA或结合位点是确定其生物功能的重要一步。一旦确定结合位点,可以使用标准的分子和生化分析进一步描述它。
此处描述的方法有两个优点。首先,它可以识别一个以前未知的结合位点,用于感兴趣的蛋白质。其次,这种方法的一个附加优势是,它同时允许饱和突变,否则,为了获得绑定站点中序列要求的可比信息,这将会是劳动密集型的。因此,它提供了一个更快,更容易,成本更低的工具,以确定RNA中的蛋白质结合位点。最初,这种方法(SELEX或通过易解体扩能对利坦的系统性进化)用于表征噬菌体T4DNA聚合酶(基因43蛋白)的结合位点,该结合位点与核糖体结合位点在其自己的mRNA中重叠。绑定站点包含一个 8 基循环序列,表示用于分析7的 65,536 个随机变体。其次,该方法还被独立用于表明,可以从大约10个13个序列8的池中选择不同染料的特定结合位点或贴合剂。事实上,这种方法已经在许多不同的环境中广泛使用,用于识别用于结合许多配体(如蛋白质、小分子和细胞)的贴合剂(RNA或DNA序列),以及催化9。例如,一个贴皮剂可以区分两种黄烷衍生物,咖啡因和茶碱,它们因咖啡因10中一个甲基组的存在而不同。我们广泛使用这种方法(SELEX或迭代选择-放大)来研究RNA结合蛋白在拼接或拼接调节11中的作用,这将是下面讨论的基础。
随机库:我们使用31个核苷酸的随机库。随机库的长度考虑松散地基于一种理念,即一般拼接因子 U2AF65绑定到分支点序列和 3' 拼接站点之间的序列。平均而言,元群中这些拼接信号之间的间距在20至40个核苷酸范围内。另一种蛋白质性致命已知结合一个不良特征的调节序列附近的3'拼接位,其目标前mRNA,变压器。因此,我们选择了31个核苷酸的随机区域,两侧是具有限制性酶位点的引物结合位点,以允许PCR扩增和附件T7RNA聚合酶启动子进行体外转录。理论库大小或复杂性为431或约1018。我们使用这个库的一小部分来准备我们的随机RNA池(#10 12-1015)用于下面描述的实验。

<table>
	<tbody>
		<tr>
			<td>Gel Electrophoresis equipment</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Plates</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Millipore</td>
			<td>HAWP</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>Schleicher &#38; Schuell</td>
			<td>PROTRAN</td>
			<td></td>
		</tr>
		<tr>
			<td>polyacrylamide gel solutions</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>NEB</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant PTB</td>
			<td>Laboratory Preparation</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse Transcriptase</td>
			<td>NEB</td>
			<td>M0277S</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum manifold</td>
			<td>Fisher Scientific</td>
			<td>XX1002500</td>
			<td>Millipore 25mm Glass Microanalysis Vacuum Filter</td>
		</tr>
		<tr>
			<td>Vacuum manifold</td>
			<td>Millipore</td>
			<td>XX2702552</td>
			<td>1225 Sampling Vacuum Manifold</td>
		</tr>
		<tr>
			<td>X-ray films</td>
			<td>Standard</td>
			<td>Standard</td>
			<td></td>
		</tr>
	</tbody>
</table>

exploring sequence space to identify binding sites for regulatory rna-binding proteins

基因调节在所有细胞中起着重要的作用。转录、转录后(或RNA处理)、翻译和翻译后步骤用于调节特定基因。序列特异性核酸结合蛋白针对特定序列来控制空间或时间基因表达。核酸中的结合位点通常以突变分析为特征。然而,许多感兴趣的蛋白质没有已知的结合位点进行这种表征。在这里,我们描述了一种识别RNA结合蛋白以前未知的结合位点的方法。它涉及从随机序列池开始的序列的迭代选择和放大。经过几轮这些步骤的转录、绑定和扩增,富集序列被序列为识别首选的绑定位点。使用体外结合测定法对这种方法的成功进行监测。随后,体外和体内功能测定可用于评估所选序列的生物相关性。这种方法允许识别和表征任何RNA结合蛋白的先前未知的结合位点,而该蛋白存在分离蛋白质结合和未结合RNA的测定。

以下观察结果表明选择放大(SELEX)是成功的。首先,我们分析了池0和选定的序列,以便与用于迭代选择-扩增方法的蛋白质结合。图2显示,哺乳动物聚丙胺-结合蛋白(PTB)几乎不能检测到结合到池0序列,但与所选序列池的亲和力较高。当我们使用比所选池相比,蛋白质浓度高出约 300 倍时,几乎无法检测到对池 0 的结合。因此,随机池或起始池与所选池之间的蛋?...

Watch this Scientific Journal Video about Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins at JoVE.com

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

探索序列空间,识别调节RNA结合蛋白的结合位点

序列特异性对基因调控至关重要。识别特定序列的调控蛋白对基因调控非常重要。为这种蛋白质定义功能结合位点是一个具有挑战性的生物学问题。此处描述了一种用于识别RNA结合蛋白结合位点的迭代方法,适用于所有RNA结合蛋白。

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

该协议用于蛋白质结合位点的饱和突变。如果您的蛋白质结合位点不知道，可以使用此协议进行识别。该技术与疾病诊断和治疗有关。我认为，它在生物医学科学方面的充分潜力仍有待充分挖掘。这种方法可以广泛应用于任何蛋白质或反应，其中所需的分子可以从不需要的分子分离。确保库正确随机化，并确保在每个结合和分离步骤中，所需分子的折叠富集性很高。它涉及几个步骤和许多细节，更好地证明，而不是用文字解释。观看此视频后，您应该能够适应如何准备随机库、合成启动的 RNA 池，并执行结合反应以分离所需的和不需要的分子，以获得高亲和力和特定的粘合剂。首先，在DNA合成器上通过化学合成合成前底和反向底剂合成。此外，合成一个随机库寡核苷酸模板与31个随机位置。在PCR管中，混合一微摩尔DNA随机库模板， 含有T7RNA聚合酶促进剂和反向底因的一微摩尔前底板，pH8下20毫摩尔三酯，1.5毫摩尔氯化镁，50毫摩尔氯化钾，每毫升1微克乙酰化牛血清白蛋白，两个单位的Taq聚合酶，和200微摩尔每个dNTP。设置 PCR 机器，具有变性、退火和扩展步骤的五个周期，然后设置一个扩展周期，将启动子连接到 DNA 库。设置含有T7转录缓冲液、单微摩尔随机库DNA、5毫摩尔DTT、2毫摩尔GTP、ATP、CTP和 UTP各1毫摩尔以及每微升2单位T7RNA聚合酶的100微升转录反应。现在戴上丙烯酸玻璃护罩和手套。通过将 5 微升或更少的 alpha-32P UTP 添加到自皮造影的转录反应中来引入放射性。在37摄氏度的微离心管中孵育反应混合物两小时。要凝胶净化RNA，请准备10%变性聚丙烯酰胺凝胶。将样品装入凝胶的孔中。将凝胶暴露在 X 射线膜中，并确定凝胶上转录本的位置并切掉凝胶片。将凝胶片放入离心管中，用均质器尖端分解成更小的片断。加入蛋白酶K缓冲液，浸入凝胶片。在室温下将管子放在螺母上至少两个小时。然后在室温下高速微离心中旋转五分钟，以清除凝胶碎屑并回收缓冲液。与上流剂、涡流和离心机一起，将同等体积的酚氯仿加入管中。再用苯酚氯仿重复提取，再用氯仿重复一次。接下来，将蛋白酶K缓冲液的水相与pH5.2、10微克tRNA或20微克糖原的1/10体积三摩尔醋酸钠混合，并在零下20摄氏度储存2至3卷乙醇。将管子在零下80摄氏度下离开一小时。在微离心中旋转含有溶液的管子5至10分钟，温度为4摄氏度。小心丢弃上清液，加入70%乙醇冲洗RNA颗粒。旋转两到五分钟。小心吸制乙醇，并风干RNA颗粒。接下来，加入50微升用DEPC处理的水，使RNA颗粒溶解。将样品留在零下20摄氏度，储存。将蛋白质和RNA结合在100微升的反应量中， 首先在含有50毫摩尔氯化钾、1毫摩尔DTT、每微升牛血清白蛋白、每微升5单位RNAsin、每微升15微克、微升tRNA和1毫摩尔EDTA时，以7微升盐酸十分法，15微克盐酸酯。盐酸十足三氯酸酯。磷酸盐，每微升TRNA，每微升5微升，每微升TRNA，1微升，1微升，1微升TRNA，1微升TRNA，1微升。1微升，每微升TRNA。1毫摩尔-12，00微升，每微升TRNA。中，每微升TRNA。1米摩尔，每微升TRNA。10微升，每微升TRNA15米。1微升，每微升TRNA。10微升，每微升TRNA。10微克，每微升tRNA，每微升TRNA15微升TRNA，每然后从适当的池中加入30微升重组蛋白PTB和10微升RNA。将含有结合反应的管子在温度块中放置约30分钟，温度为25摄氏度。接下来，通过四轮选择和扩增，从未绑定RNA中对绑定RNA进行分馏。首先，通过连接到真空歧管的硝基纤维素过滤器，在室温下过滤100微升样品。RNA-蛋白质复合物仍保留在过滤器上。用无菌剃须刀刀片，将过滤器切成碎片，然后插入离心管。将滤片浸入蛋白酶K缓冲液中，并放在滚筒上至少三个小时或过夜以回收RNA。要使RNA样品脱蛋白，加入同等体积的酚-氯仿、涡流，然后在室温下高速离心五分钟。获得水相，并再次提取氯仿。之后，将上经剂与1/10体积的三摩尔醋酸钠混合在pH 5.2和2至3卷绝对乙醇中。将管子放在零下80摄氏度的冰柜中30分钟，然后高速离心10分钟。使用 70% 乙醇重复洗涤和干燥步骤，并在 DEPC 处理的水中溶解 RNA。第一轮过滤结合测定后，进行三轮。接下来，执行两轮转录、结合和扩增，将蛋白结合的RNA分数与未结合的馏分分离。在 5 倍 TBE 缓冲液中预制一种原生 5%聚丙烯酰胺凝胶，丙烯酰胺比为 60 比 1。然后建立RNA蛋白结合反应。将凝胶放在充满 5x TBE 缓冲液的电泳装置中，温度为 4 摄氏度。应用250伏特15分钟，将结合反应移液到不同的井中。然后，通过以250伏电压运行电泳一到两个小时，进一步从未绑定RNA中对绑定RNA进行分馏。接下来，将凝胶暴露在 X 射线膜中，并使用自扫描确定绑定 RNA 的位置。用绑定的RNA切掉凝胶片，并将其插入管中。粉碎凝胶片，在蛋白酶K缓冲液中孵育三小时或过夜。如前面所述，使用苯酚氯仿和氯仿重复萃取。从溶解的RNA合成cDNA。准备20微升的反应，包括两个10倍RT缓冲液微升，2个MV逆转录酶微升，一个反素转微升，5微升水，10微升溶解RNA。在42摄氏度下孵育60分钟。如前所述，使用 20 到 25 个 PCR 周期放大 cDNA。重复RNA合成、蛋白质结合和蛋白质结合和未结合分数分离的过程。要分析蛋白质结合，请使用凝胶移动移位测定或过滤器结合测定来确定每个池内选定池或单个序列的结合亲和力和特异性。使用自动测量或荧光成像仪检测和量化绑定分数和未绑定分数中的波段。使用克隆和序列对齐继续协议。在这项研究中，成功地实现了选择放大。具有300倍高蛋白浓度的哺乳动物多酰胺-二聚酰胺结合蛋白显示，与池零序列几乎检测不拟结合，但对所选序列池的相关性很高。选择扩增成功识别在RNA结合蛋白与首选或共识结合位点。添加与上游三等分线拼接站点绑定的重组 PTB 会导致替代或下游三等拼接站点的激活。相比之下，如果重新组合的 hnRNP C 导致对两个三个黄金拼接站点的压制。添加重组一般拼接因子 U2AF65 扭转了 hnRNP C 中压三个黄金拼接站点的压制。重要的是要记住，一个好的随机库和适当的绑定条件，在每个绑定步骤给予高折叠丰富是必要的成功。适当的功能测定和体内测试可用于验证此方法的结果。在基因表达领域，利用该技术对多种蛋白质的功能进行了研究。使用聚丙烯酰胺和放射性时，使用手套和放射性密封进行保护非常重要。

Research

JoVE Journal

Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

科研

遗传学

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

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 ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

逆转录病毒扫描：映射MLV集成站点以定义细胞特异性监管区域

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

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

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 ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...