Name: complementation of splicing activity by a galectin-3 - u1 snrnp complex on beads
Uploaded: 2020-12-09T13:00:00
Description: Watch this Scientific Journal Video about Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads at JoVE.com

这项工作得到了美国国家科学基金会拨款MCB-0092919和密歇根州立大学校内研究拨款09-CDFP-2001（到RJP）以及美国国立卫生研究院拨款GM-38740和密歇根AgBioResearch项目MICL02455（到JLW）的支持。
剪接测定中使用的MINX前mRNA底物是Susan Berget博士（美国德克萨斯州休斯顿贝勒医学院）的亲切礼物。

1. 关于一般程序的说明
<ol><li>确保所有化学物质（缓冲液成分，酶等）均不含核糖核酸酶（RNase）。隔离所有商业购买的试剂瓶，使其免受一般实验室使用。在实验过程的所有步骤中都要戴上手套。仅使用已经烘烤的玻璃器皿和器皿（见下面的步骤1.2）和经过预处理的溶液（见下面的步骤1.3）。</li>
	<li>在177°C下烘烤所有玻璃器皿（烧杯，烧瓶，瓶子，移液器等）至少4小时。 在相同条件下烘烤?...

<ol>
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本报告提供了实验细节，记录了被困在抗Gal3包被珠上的Gal3 - U1 snRNP复合物可以与前mRNA底物结合，并且这种三元复合物可以恢复U1 snRNP耗尽的NE的剪接活性。.早期的免疫荧光和亚细胞分馏研究提供了 Gal3 与剪接机制组分关联的初步线索：在核斑点中与 snRNPs 的 Sm 核多肽以及富含丝氨酸和精氨酸的 （SR） 家族的调味因子24，25 进行共定位?...

大多数真核信使RNA（mRNA）的产生涉及在称为前mRNA剪接的核过程中去除内含子和连接外显子1。两类RNA-蛋白质复合物（RNPs）通过剪接体复合物指导前信使RNA加工成成熟的mRNA。一类是新生的前信使RNPs，由异质核RNP蛋白和其他RNA结合蛋白（包括SR家族的一些成员）结合共转录形成，产生hnRNP复合物2。第二类富含尿嘧啶的小核RNPs（U snRNPs与U1，U2，U4，U5和U6 snRNA）与U特异性和核心蛋白相关3，4。U snRNPs以有序的方式与信使前RNP的特定区域在动态重塑途径中相互作用，因为内含子被切除并且外显子被连接以产生成熟的mRNPs5。许多其他核蛋白参与这些处理事件6。
如耗竭-重建研究所示，半乳糖凝集素-1（Gal1）和半乳糖凝集素-3（Gal3）是剪接途径中必需因子的两种蛋白质7，8。从剪接中去除两种半乳糖凝集素，可在早期阶段消除剪接体组装和剪接活性。将任一半凝集素添加到这种双耗竭的NE中可以恢复两种活性。Gal1 和 Gal3 是活性剪接体的组分，其前 mRNA 的特异性免疫沉淀、剪接中间体和成熟 mRNA 通过 Gal1 或 Gal39 特异性抗血清证明了这一点。重要的是，Gal3与剪接途径外的NE中含有的内源性U snRNA结合，如抗Gal3抗血清沉淀snRNPs所示10。最后，HeLa细胞中Gal3的沉默改变了许多基因的剪接模式11。
在预孵育以拆卸预制的剪接体12的NE中，snRNPs存在于从7S到大于60S的甘油梯度沉淀的多个复合物中。虽然甘油梯度分馏是分离剪接体复合物和组分的常用技术（例如参见参考文献13，14，15 ），但我们通过使用抗体免疫沉淀表征特定组分来扩展该方法。在10S下沉淀的snRNP仅含有U1 snRNA和Gal3。10S级分的免疫沉淀与对Gal3或U1 snRNP特异性的抗血清共沉淀U1和Gal3均表明一些U1 snRNP单颗粒与Gal310结合。由于U1 snRNP是第一个与剪接体组装中的前mRNP结合的复合物1，5，因此该步骤代表了Gal3进入剪接途径的潜在入口位点。在此基础上，我们表明与含有抗Gal3的珠子结合的10S Gal3-U1 snRNP单颗粒恢复了U1 snRNP耗尽的NE的剪接活性，将该复合物建立为Gal3被招募到剪接体途径中的一种机制16。这与在剪接反应的特定阶段分离剪接体并编目相关因子的尝试形成鲜明对比17，18。在此类研究中，确定了某些因素在某个时间点的存在，但不确定它们被加载的机制。
我们之前在关于半乳糖凝集素在前mRNA剪接中的作用的文献中详细描述了NE的制备，剪接底物，剪接反应混合物的组装以及产物的分析19。我们现在描述了核提取物的分馏以获得富含Gal3 - U1 snRNP复合物的馏分以及后一种复合物的免疫选择以在U1耗尽的核提取物中重建剪接活性的实验程序。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61990/61990fig01.jpg"> 图1：示意图，说明在珠子上用Gal3-U1 snRNP复合物耗尽U1 snRNP的核提取物中剪接活性的互补性。未结合的分数耗尽U1 snRNP（U1ΔNE）。（B）缓冲液D（NE（D））中的NE通过超速在12%-32%的甘油梯度上分馏。将对应于10S区域的馏分（级分3-5）与与抗Gal3抗体（αGal3微球）共价偶联的微球混合。与磁珠结合的材料含有Gal3-U1 snRNP单颗粒。（C）使用32P标记的MINX前mRNA底物在剪接测定中将来自（B）部分的Gal3-U1 snRNP配合物与来自（A）部分的U1ΔNE混合，并通过凝胶电泳和放射自显像分析剪接反应的中间体和产物。<a href="https://www.jove.com/files/ftp_upload/61990/61990fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-U1 snRNP</td>
			<td>The Binding Site</td>
			<td>Hu ENA-RNP #33471</td>
			<td>human autoimmune serum specific for U1 snRNP</td>
		</tr>
		<tr>
			<td>bottle top vacuum filter</td>
			<td>Fisher Scientific</td>
			<td>Corning 431153 (0.22 &#956;m; PES 150 ml)</td>
			<td>for filtering solutions containing Tris</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>International Equipment Company</td>
			<td>IEC Model PR-6</td>
			<td>for pelletting Sepharose beads in immunoprecipitation</td>
		</tr>
		<tr>
			<td>diethylpyrocarbonate (DEPC)</td>
			<td>Sigma-Aldrich</td>
			<td>159220-5G</td>
			<td>for treatment of water used in preparation of all solutions</td>
		</tr>
		<tr>
			<td>dimethylpimelimidate (DMP)</td>
			<td>Sigma-Aldrich</td>
			<td>80490-5G</td>
			<td>for cross-linking antibody to Sepharose beads</td>
		</tr>
		<tr>
			<td>electrophoresis cell</td>
			<td>BioRad Laboratories, Inc</td>
			<td>Mini-Protean II</td>
			<td>for SDS-PAGE separation of proteins</td>
		</tr>
		<tr>
			<td>ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>411000-100ml</td>
			<td>for blocking after the cross-linking reaction</td>
		</tr>
		<tr>
			<td>gel electrophoresis system</td>
			<td>Hoefer, Inc</td>
			<td>HSI SE 500 Series</td>
			<td>for separating snRNAs by gel electrophoresis</td>
		</tr>
		<tr>
			<td>gel slab dryer</td>
			<td>BioRad</td>
			<td>Model 224</td>
			<td>for drying gel slabs for autoradiography</td>
		</tr>
		<tr>
			<td>Hybond ECL membrane</td>
			<td>GE Healthcare</td>
			<td>RPN3032D (0.2 &#956;m; 30 cm x 3 m)</td>
			<td>for immunoblotting of proteins on membrane</td>
		</tr>
		<tr>
			<td>microdialyzer (12 x 100 &#956;l sample capacity)</td>
			<td>Pierce</td>
			<td>Microdialyzer System 100</td>
			<td>for exchanging the buffer of nuclear extract&#160;&#160;</td>
		</tr>
		<tr>
			<td>microdialyzer membranes (8K cutoff)</td>
			<td>Pierce</td>
			<td>66310</td>
			<td>for exchanging the buffer of nuclear extract&#160;</td>
		</tr>
		<tr>
			<td>non-fat dry milk</td>
			<td>Spartan Stores</td>
			<td>Spartan Instant Non-fat Dry Milk</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A Sepharose CL-4B</td>
			<td>Millipore-Sigma</td>
			<td>GE 17-0780-01</td>
			<td>for coupling antibody to beads</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Millipore-Sigma</td>
			<td>P2308-5mg</td>
			<td>for stopping the splicing reaction to isolate the RNAs</td>
		</tr>
		<tr>
			<td>RNasin</td>
			<td>Promega</td>
			<td>N2111</td>
			<td>for inhibiting ribonuclease activity</td>
		</tr>
		<tr>
			<td>rocker/rotator</td>
			<td>Lab Industries, Inc&#160;</td>
			<td>Labquake Shaker 400-110</td>
			<td>for mixing protein solutions in coupling reactions and in immunoprecipitation</td>
		</tr>
		<tr>
			<td>Safety-Solve</td>
			<td>Research Products International Corp.</td>
			<td>No. 111177</td>
			<td>scintillation counting cocktail for determination of radioactivity in splicing substrate</td>
		</tr>
		<tr>
			<td>scintillation counter</td>
			<td>Beckman Instruments</td>
			<td>LS6000SC</td>
			<td>scintillation counter for determination of radioactivity&#160;</td>
		</tr>
		<tr>
			<td>speed vaccum concentrator</td>
			<td>Savant</td>
			<td>SVC 100H</td>
			<td>for drying ethanol-precipitated RNA pellets</td>
		</tr>
		<tr>
			<td>Transphor electrophoresis unit</td>
			<td>Hoefer, Inc</td>
			<td>Hoefer TE Series Transphor</td>
			<td>for protein transfer from SDS-PAGE to blotting membrane</td>
		</tr>
	</tbody>
</table>

complementation of splicing activity by a galectin-3 - u1 snrnp complex on beads

经典的耗竭重建实验表明，半乳糖凝集素-3是核提取物中必需的剪接因子。本文探讨了半乳糖凝集素-3掺入剪接途径的机制。HeLa细胞核提取物在12%-32%的甘油梯度上沉淀产生富含含有半乳糖凝集素-3和U1 snRNP的内源性〜10S颗粒的馏分。我们现在描述了一种方案，用于消耗U1 snRNP的核提取物，同时伴随剪接活性的丧失。U1耗尽提取物中的剪接活性可以通过捕获在琼脂糖珠上的半乳糖凝集素-3 -U1 snRNP颗粒与抗半乳糖凝集素-3抗体共价偶联来重建。结果表明，半乳糖凝集素-3 - U1 snRNP - 前mRNA三元复合物是导致剪接反应中间体和产物的功能性E复合物，半乳糖凝集素-3通过其与U1 snRNP的结合进入剪接途径。使用在磁珠上选择的复合物亲和力或免疫力来重建特定剪接因子耗尽的提取物中的剪接活性的方案通常可能适用于其他系统。

将U1 snRNP（U1ΔNE来自2.2.6节）和来自抗Gal3免疫沉淀的甘油梯度10S区域的Gal3 -U1 snRNP复合物的NE耗尽（步骤3.2.7）在剪接反应中混合。该反应混合物含有U1 snRNA（图2A，泳道3）以及U1特异性蛋白U1-70K（图2B，泳道3）。正如预期的那样，反Gal3沉淀了Gal3（图2B，通道3）。这些组分（U1 snRNA，U1-70K蛋白和Gal3）在免疫?...

Watch this Scientific Journal Video about Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads at JoVE.com

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

本文描述了（a）核提取物中U1 snRNP的消耗，伴随剪接活性的丧失;（b）通过与珠子共价偶联的微乳凝集素-3 - U1 snRNP颗粒与抗半乳糖凝集素-3抗体共价结合，在U1耗尽的提取物中重建剪接活性。

半乳糖凝集素-3 - U1 snRNP复合物在珠子上的剪接活性的补充

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

Research

JoVE Journal

Biochemistry

A Fluorescence-based Assay of Phospholipid Scramblase Activity

磷脂Scramblase活动的基于荧光的测定法

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

科研

生物化学

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

巨囊泡封装微球通过一个水包油乳液的离心制备

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

镍 - 席夫碱的水解复杂环境条件适宜酸不稳定保护基团的保留

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

酶的结晶和结构确定：多功能微流体芯片中串行晶体学的基板复合物

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

测量CD95死亡诱导信号传导复合物的组成和该复合物中Procaspase-8的处理

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...