Name: isolation of cognate rna-protein complexes from cells using oligonucleotide-directed elution
Uploaded: 2017-01-16T12:30:00.000+00:00
Duration: 10 min 53 s
Description: Watch this Scientific Journal Video about Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution at JoVE.com

作者非常感谢由美国国立卫生研究院P50GM103297，P30CA100730和综合癌症P01CA16058支持。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 缓冲成分 </td></tr><tr><td> 洗涤液: </td></tr><tr><td>的50mM的Tris-HCl，pH 7.4的</td></tr><tr><td> 150毫米氯化钠</td></tr><tr><td> 3毫米氯化镁</td></tr><tr><td> 低盐缓冲液: </td></tr><tr><td>的20mM的Tris-HCl，pH值7.5 </td></tr><tr><td> 10毫米氯化钠</td></tr><tr><td> 3毫米氯化镁</td></tr><tr><td> 2毫摩尔DTT </td></tr><tr><td> 1×蛋白酶抑制剂鸡尾酒无EDTA </td></tr><tr><td>核糖核酸酶出5微升/毫升（RNA酶抑制剂） </td></tr><tr><td> 细胞质裂解液: </td></tr><tr><td> 0.2M的蔗糖</td></tr><tr><td> 1.2％的Triton X-100 </td></tr><tr><td> NETN-150洗涤液: </td></tr><tr><td>的20mM的Tris-HCl，pH 7.4的</td></tr><tr><td> 150毫米氯化钠</td></tr><tr><td> 0.5％的NP-40 </td></tr><tr><td> 3毫米氯化镁</td></tr><tr><td> 10％甘油</td></tr><tr><td> 结合缓冲液: </td></tr><tr><td>的10mM HEPES pH 7.6的</td></tr><tr><td> 40毫米氯化钾</td></tr><tr><td> 3毫米氯化镁</td></tr><tr><td> 2毫摩尔DTT </td></tr><tr><td> 5％甘油</td></tr></tbody></table> 表2:缓冲组合物。 1.细胞及亲和基质的制备<ol><li>培养的兴趣在10cm皿分汇合（80％）的细胞系。使用了FLAG标签NLS-MS2外壳蛋白的免疫沉淀无关（IP）10厘米板。执行使用抗血清的FLAG表位标签的IP。当表达FLAG标签的MS2外壳蛋白质粒转染细胞24-48小时提前收获20。 注:特定的RNP可以从核或细胞质中富集集成电路裂解物或生物化学分级分离制剂，如蔗糖梯度的级分。建议收获来自非转染细胞的裂解物在平行于提起附加阴性对照。 </li><li>每转的G蛋白磁珠浆IP 60微升至1.7 ml离心管。 </li><li>放置在磁铁架的管离心管到珠子从存储溶液中分离出来。 </li><li>用微量精心绘制，将其取出存储解决方案。 </li><li>从磁体机架上卸下管。 </li><li>洗涤和平衡1×洗涤缓冲液600微升珠（珠的使用量的10倍）（20毫摩尔Tris盐酸，pH为7.4; 3毫MgCl 2的;以及150mM的氯化钠）和最终过端3旋转分钟，在室温。 </li><li>放置在磁铁架管和除去洗涤缓冲液。 </li><li> 1×洗涤缓冲液和免疫沉淀FLAG抗体的10体积（600微升）添加到平衡的公关oteinģ磁珠（根据由生产用于免疫沉淀推荐的量）为至少30分钟至缀合的免疫沉淀抗体和旋转终端过端在室温下。使用相应的同种型IgG作为一个合适的抗体的阴性对照。 </li><li>放置在磁铁架的管收集珠子并除去上清液。 </li><li>从磁体机架上卸下管中，用1×洗涤缓冲液和旋转的10体积（600微升）洗抗体缀合的小珠在室温下3分钟。重复此步骤两次。 </li><li>放置在磁铁架管和除去洗涤缓冲液。 </li></ol> 2.收获的RNP 注:在步骤1.8之后的孵化时间准备的RNP。 <ol><li>通过抽吸除去从细胞培养基中并用1-5毫升冰冷的1X磷酸盐缓冲盐水（PBS）洗细胞两次。使用细胞刮赶走潮湿，在4℃收集前贴壁细胞通过在226×g离心4分钟离心。 </li><li>添加375微升冰冷却的，低盐缓冲液（20mM的Tris-HCl，pH值7.5; 3毫MgCl 2的10毫摩尔NaCl; 2mM的DTT; 1×蛋白酶抑制剂鸡尾酒，无EDTA;和5μl/ ml的RNA酶抑制剂）的细胞沉淀，并允许通过将它放置在冰上5分钟肿胀。 注:裁缝根据细胞沉淀的大小低盐缓冲液体积。对于从10cm平板1.2×10 6个细胞，375微升缓冲液中是足够的。 </li><li>收集胞质细胞裂解物中，添加125微升冰冷的裂解缓冲液（0.2M蔗糖/ 1.2％的Triton X-100），然后执行10笔用Dounce匀浆，这是在一个冰桶预冷。 注意:为了收集总细胞提取物，被推荐用于增溶的核质/染色质标准的RIPA裂解缓冲液。 </li><li>自旋以最快的速度（16,000 XG）1分钟微量;这将清除碎片的上清液第二核。 </li><li>将上清转移到新的1.7 ml离心管，其已经在冰上。确定由一个标准，实验室优选的方法，如Bradford测定23的总蛋白浓度。保留至少10％的等分试样用于蛋白质印迹分析，以用作一个输入控制。立即使用收获的细胞裂解物进行免疫沉淀或储存在-80℃以供将来分析。 注:根据我们的经验，这些样品可以存储和几个月后用于免疫分析没有诚信的一种妥协。 </li></ol> 3.免疫沉淀<ol><li>添加收获细胞裂解物的所需体积，基于在步骤2.5中确定的蛋白浓度，对目标抗体缀合的小珠。用1×洗涤缓冲液，使总体积达600微升和旋转终端过端在室温下90分钟。 注:该时间周期足以产生高亲和性的RNP复合体的健壮隔离，同时最小化非特异性结合。稀替代RNP来源，如蔗糖梯度的级分，至少1:1与1×洗涤缓冲液，然后用预先制备的珠 - 抗体复合孵育它们，如上所述。 </li><li> 90分钟温育后，放置在磁铁架知识产权管收集珠子。保留上清液作为流通。 注:此第一流通是衡量IP效率的重要对照样品。免疫印迹测定法将提供的IP效率的指示，以及所研究的相互作用的特异性。它建议保留该上清液用于下游分析。 </li><li> 150毫摩尔NaCl; 3毫MgCl 2的0.5％NP40;用冰冷的NETN-150洗涤缓冲液（20mM的Tris-HCl，pH值7.4的10体积（600微升）洗RNP结合，抗体缀合的小珠和10％甘油）根据在室温下3分钟的旋转。重复步骤3次。 注:此缓冲区从裂解缓冲液的不同，有效地降低了弱的或非特异性的关联。 </li><li>以下最后的洗涤，悬浮固定化的RNP复合物在60微升冰冷的1X结合缓冲液（10mM HEPES，pH值7.6; 40毫氯化钾; 3毫米氯化镁2; 5％甘油;和2mM DTT）中，并储备10％对于Western印迹和RNA分离20％的固定的复合物珠粒。 </li></ol> 4.洗脱<ol><li>调整剩余体积至100μl用冰冷的1X结合缓冲液，并在70℃下加热管3分钟。 </li><li>以分离同源RNA-蛋白质复合物，添加〜30-nt的DNA寡核苷酸是与感兴趣的RNA的3&#39;序列的边界互补并在室温下在温和温育30分钟摇动（100nM的寡核苷酸是足够了）。 注:寡核苷酸是反义邻近于翻译的核苷酸残基开始现场用作为例四氯乙烯构建体E在此协议。适当的序列互补性是由〜40％的GC含量提供。设计用于高效杂交与靶RNA的最小互补区域的反义寡核苷酸。 </li><li>添加5-10单位RNA酶H的该管，在室温下孵育1小时以裂解RNA的RNA:DNA杂交体。将上清转移到无菌1.7毫升离心管中;该样品中含有的利益所捕获的RNP复合物。 注:根据同源RNA的可获得性，两个或更多个轮RNA酶H处理可能是有用的，以增加样品丰度为下游分析。 </li><li>用洗脱液为20％的Western印迹已知相关蛋白并随后通过RT qPCR的洗脱液用于RNA分离的20％。剩下的60％可以用于质谱或鉴定蛋白质组分。 </li><li>平行地，若使保留细胞裂解物对RNA分离和RT-PCR分析的等分试样。这ASSEssment提供RNP园区内的RNA富集的迹象。 注:使用在控制免疫印迹分离蛋白制剂，以确定该核糖核酸酶H裂解能有效地从immunuoprecipitated复杂释放RNP。 </li></ol> 5.蛋白电泳和Western blot分析<ol><li>根据标准的实验室协议受试者的总样品进行SDS-PAGE和免疫印迹分析的约10-20％。 注意:此步骤用于之前下游RNA分析验证的IP效率和特异性。它也可以用来评估所述分离的RNP复合体的蛋白质组合物。 </li></ol> 6.收集免疫沉淀的RNA 注:RNA分离可通过Trizol法或以下所描述的协议来实现。 <ol><li>在750微升异硫氰酸胍试剂样品总数的一半悬浮和孵化在房T5分钟提取从捕获的PCE-RNP复合物的RNA之前emperature。 </li><li>加入200μl的氯仿至管，剧烈振荡10秒，并在室温下孵育3分钟。 </li><li>在4℃下16000×g离心15分钟离心后，收集水相到重新管和添加异丙醇等体积。拌匀并在室温下孵育至少10分钟。加入1μl乙二醇蓝色的样品，并在冷冻高效沉淀或加工-20℃存放在未来某一日期。 </li><li>离心反应管以16,000 xg离心在4℃下10分钟。小心收集并弃去上清液，以便不干扰RNA沉淀;建议使用的p-200微管尖端，以促进该过程。 </li><li> 16,000 xg离心加入500μl75％乙醇到每个管中，涡旋，离心管5分钟，在4℃下。认真收集并弃去上清液按步骤6.4。空气-干燥在100μl的无RNase水2-3分钟，重悬沉淀。 注意:不要延长时间沉淀晾晒，以避免与悬浮的一个问题。 </li><li>应用100微升RNA样品的RNA清理柱。使用制造商的协议处理。洗脱在无RNase水和存储的30微升的RNA在-80℃长达3个月。 注意:此分离的RNA适合于通过RT-实时PCR（qPCR的），微阵列，和RNA测序下游分析。取决于丰RNP和与感兴趣的RNP分离效率，相同的裂解物可以经受两个或更多轮的IP，以产生适用于下游分析足够的RNA。 </li></ol> 7. RNA反转录和扩增的cDNA通过PCR <ol><li>若使分离的RNA通过用高质量的逆转录酶（RT）的随机引物反转录，根据厂家专业cturer的说明。 </li><li>为了扩增目的RNA，设计核糖核酸酶H裂解序列内的特定基因的反义引物。使用反义寡核苷酸，随机引物或寡-dT引物相似量的（见材料表 ）。 注:寡聚-dT引物和聚腺苷酸化的mRNA提供用于RT-PCR反应阳性对照反应。 </li><li>储备RT反应（1微升）用于制备的qPCR串联做阴性对照溶胞产物的IP和阳性对照DNA样本来定义截止和产生的标准曲线的5％。如果制备的qPCR的CT值是超出标准曲线的范围内，稀释在1个连续的RT反应:5稀释液，并重复步骤7.2。 注:对于RNA测序和质谱技术，请参阅补充方法文件。 <a href="http://ecsource.jove.com/files/ftp_upload/54391/Supplementary_method_File_Jove.docx" target="_blank">请点击此处Ť</a>Ø查看本补充方法文件。 （右键点击下载）。 </li></ol>

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Virol. 73 (6), 4847-4855 (1999).</li><li>Bolinger, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+helicase+A+interacts+with+divergent+lymphotropic+retroviruses+and+promotes+translation+of+human+T-cell+leukemia+virus+type+1.">RNA helicase A interacts with divergent lymphotropic retroviruses and promotes translation of human T-cell leukemia virus type 1.</a> Nucleic Acids Res. 35 (8), 2629-2642 (2007).</li><li>Bolinger, C., Sharma, A., Singh, D., Yu, L., Boris-Lawrie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+helicase+A+modulates+translation+of+HIV-1+and+infectivity+of+progeny+virions.">RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions.</a> Nucleic Acids Res. 38 (5), 1686-1696 (2010).</li><li>Lee, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+the+DHX9+helicase+induces+premature+senescence+in+human+diploid+fibroblasts+in+a+p53-dependent+manner.">Suppression of the DHX9 helicase induces premature senescence in human diploid fibroblasts in a p53-dependent manner.</a> J.Biol.Chem. 289 (33), 22798-22814 (2014).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal.Biochem. 72, 248-254 (1976).</li><li>Glenn, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+protein+samples+for+mass+spectrometry+and+N-terminal+sequencing.">Preparation of protein samples for mass spectrometry and N-terminal sequencing.</a> Methods Enzymol. 536, 27-44 (2014).</li><li>Keene, J. D., Komisarow, J. M., Friedersdorf, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIP-Chip:+the+isolation+and+identification+of+mRNAs,+microRNAs+and+protein+components+of+ribonucleoprotein+complexes+from+cell+extracts.">RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts.</a> Nat.Protoc. 1 (1), 302-307 (2006).</li><li>Ranji, A., Shkriabai, N., Kvaratskhelia, M., Musier-Forsyth, K., Boris-Lawrie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+double-stranded+RNA-binding+domains+of+RNA+helicase+A+are+necessary+for+selective+recognition+and+translation+of+complex+mRNAs.">Features of double-stranded RNA-binding domains of RNA helicase A are necessary for selective recognition and translation of complex mRNAs.</a> J.Biol.Chem. 286 (7), 5328-5337 (2011).</li></ol>

The authors have nothing to disclose.

这里描述的RNP隔离和同源RNA识别策略是调查一个特定的RNA蛋白质相互作用，发现候选蛋白共同调节细胞的特异性RNP的选择性手段。 使用寡核苷酸定向RNA酶H切割以分离的RNP的优点是捕捉和具体分析过下游结合到感兴趣的顺式作用RNA元件异质的RNP的RNP顺式作用RNA元件的能力。因为在给定的RNP同源RNA的丰度是无RNA酶H裂解分离所收集的RNP的一小部分，该工作流的主要缺点是同源的RNP...

转录后基因表达被精确调节，以在细胞核中的DNA的转录开始。通过RNA结合蛋白（限制性商业惯例）控制，基因生物合成及代谢发生在高度动态的核糖核蛋白颗粒（体RNP），其联营公司及RNA代谢1-3的进展过程中与底物前体mRNA的解离。在RNP部件的动态变化影响的mRNA的转录后命运和初级成绩单，它们的核运输和定位，它们作为mRNA翻译模板活动，和成熟的mRNA的最终成交的处理过程中提供质量保证。 许多蛋白质由于其保守的氨基酸结构域，包括RNA识别基序（RRM）的指定为限制性商业惯例中，双链RNA结合结构域（RBD），和碱性残基的延伸（ 例如，精氨酸，赖氨酸，和甘氨酸） 4。限制性商业惯例通常是通过免疫策略分离和筛选，以确定其同源的RNA。某些限制性商业惯例共调节预的mRNA即在功能上相关的，被指定为RNA的调节子5-8。这些限制性商业惯例，其同源的mRNA，有时非编码RNA，形成催化的RNP在组成而改变;其独特之处是由于相关因素的各种组合，以及以时间顺序，位置，以及它们之间的相互作用9的持续时间。 RNA免疫沉淀（RIP）是一个强大的技术来隔离细胞的RNP，并确定使用序列分析10-13相关的成绩单。从候选移动到全基因组筛选是通过RIP与微阵列分析14或高通量测序（RNA测序）15组合是可行的。同样地，共沉淀的蛋白质可通过质谱法鉴定，如果它们足够丰富和可分离来自共沉淀的抗体16，17。在这里，我们解决了分离从培养的人类细胞的特定的同源RNA的RNP部件的方法，虽然该方法是可改变的植物细胞，真菌，病毒和细菌的可溶性裂解物。该材料的下游分析包括候选识别和验证通过免疫印迹，质谱法，生化酶促测定法，RT-qPCR的，微阵列，和RNA测序，如总结于图1。 定的RNP在在转录后水平控制基因表达的基本作用，在组分限制性商业惯例或它们的可访问的表达的改变，以同源的RNA可能是有害的细胞，并与几种类型的疾病，包括神经系统疾病18相关联。 DHX9 / RNA解旋酶A（RHA）是必要的细胞和逆转录病毒起源6选定的mRNA的翻译。这些同源的RNA具有内结构相关的顺式作用元件的5&#39;端非编码区，其被指定为转录后控制元件（PCE）19。 RHA-PCE活性是必需的许多逆转录病毒，包括HIV-1的有效帽依赖性翻译，和生长调节基因，包括JUND 6,20,21。由必需基因（dhx9）编码，RHA是细胞增殖和其下调基本消除了细胞活力22。 RHA-PCE的RNP的分子分析对于理解为什么RHA-PCE活性是必要的，以控制细胞增殖的重要步骤。 在稳定状态下或在细胞的生理扰动RHA-PCE RNP组分的确切性质要求RHA-PCE的RNP在足够丰富用于下游分析选择性富集和捕获。这里，逆转录病毒PCEgag的RNA具有标记为开放阅读框架内的MS2外壳蛋白（CP）的顺式作用的RNA结合位点的6份。该MS2外壳蛋白外切genously共表达质粒转染PCEgag的RNA以促进生长的细胞的RNP组件。含有同源MS2标记PCEgag RNA中的MS2外壳蛋白的RNP从细胞提取物中免疫沉淀和磁珠（ 图2a）捕获。选择性地捕获结合于四氯乙烯的RNP部件，固定RNP用至远端的PCE序列互补的寡核苷酸温育，形成的RNA-DNA杂交是对RNA酶H活性的底物。因为四氯乙烯是位于非翻译区的5&#39;5的终端"，寡核苷酸是相邻的逆转录病毒翻译起始位点（的gag起始密码子）的RNA序列互补。邻近在gag起始密码子从固定化的RNP，将其收集作为洗脱剂释放的5&#39;非编码区复杂RNA酶H裂解。此后，将样品通过RT-PCR评估，以证实PCEgag的，并通过SDS PAGE和免疫印迹捕获确认captu重新目标MS2外壳蛋白。四氯乙烯相关的RNA结合蛋白的验证，DHX9 / RNA解旋酶A，然后进行。

<table><tbody><tr><td>Dynabeads Protein A</td><td>Invitrogen</td><td>10002D</td><td></td></tr><tr><td>Dynabeads Protein G</td><td>Invitrogen</td><td>10004D</td><td></td></tr><tr><td>Anti-FLAG antibody</td><td>Sigma</td><td>F3165</td><td></td></tr><tr><td>Anti-FLAG antibody</td><td>Sigma</td><td>F7425</td><td></td></tr><tr><td>Anti-RHA antibody</td><td>Vaxron</td><td>PA-001</td><td></td></tr><tr><td>TRizol LS reagent</td><td>Life technology</td><td>10296-028</td><td></td></tr><tr><td>RNase H</td><td>Ambion</td><td>AM2292</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>BP1145-1</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>BP26184</td><td></td></tr><tr><td>RNaeasy clean-up column</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>Omniscript reverse transcriptase</td><td>Qiagen</td><td>205113</td><td></td></tr><tr><td>RNase Out</td><td>Invitrogen</td><td>10777-019</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche</td><td>5056489001</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>NP-40</td><td>Sigma</td><td>98379</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>17904</td><td></td></tr><tr><td>Random hexamer primers</td><td>Invitrogen</td><td>N8080127</td><td></td></tr><tr><td>Oligo-dT primers</td><td>Invitrogen</td><td>AM5730G</td><td></td></tr><tr><td>PCR primers</td><td>IDT</td><td></td><td>Gene specific primers for&amp;nbsp; PCR amplification</td></tr><tr><td>Oligonucleotide for RNase H mediated cleavage</td><td>IDT</td><td></td><td>Anti-sense primer for target RNA</td></tr><tr><td>Trypsin</td><td>Gibco Life technology</td><td>25300-054</td><td></td></tr><tr><td>DMEM tissue culture medium</td><td>Gibco Life technology</td><td>11965-092</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco Life technology</td><td>10082-147</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-5</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>S642-212</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher Scientific</td><td>BP214</td><td></td></tr><tr><td>DTT</td><td>Fisher Scientific</td><td>R0862</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>BP220-212</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Bio-Rad</td><td>1620112</td><td></td></tr><tr><td>Magnetic stand</td><td></td><td></td><td>1.7 ml micro-centrifuge tube holding</td></tr><tr><td>Laminar hood</td><td></td><td></td><td>For animal tissue culture</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td>For animal tissue culture</td></tr><tr><td>Protein gel apparatus</td><td></td><td></td><td>Protein sample separation</td></tr><tr><td>Protein transfer apparatus</td><td></td><td></td><td>Protein sample transfer</td></tr><tr><td>Ready to use protein gels (4-15%)</td><td></td><td></td><td>Protein sample separation</td></tr><tr><td>Table top centrifuge</td><td></td><td></td><td>Pellet down the sample</td></tr><tr><td>Table top rotator</td><td></td><td></td><td>Mix the sample end to end</td></tr><tr><td>Vortex</td><td></td><td></td><td>Mix the samples</td></tr></tbody></table>

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.

RIP前鉴定结果逆转录病毒插科打诨RNA和选择的共沉淀的细胞的RNA与DHX9 / RHA，包括HIV-1 6，勇得6户珥（弗里茨和鲍里斯-劳列，未发表）。反转录病毒的5&#39;非编码区已被证明与DHX9 / RHA在细胞核共沉淀并在多核糖体的细胞质共同隔离。它是唯一定义为顺式短效后转录控制元件（PCE）6。为了分离形成增殖细胞PCEgag的RNA核糖核蛋白RHA复合物（体RNP...

Watch this Scientific Journal Video about Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution at JoVE.com

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

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. ...

isolation-cognate-rna-protein-complexes-from-cells-using

Research

JoVE Journal

Biochemistry

9.0K 次观看.  University of Minnesota. 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. 

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

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

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 ...

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

生物化学

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

生物素和光裂解链接器所附 rna 对高分子配合物的单步纯化

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

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 ...

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 ...

生物学

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

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.

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

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