Name: novel rna-binding proteins isolation by the rapid methodology
Uploaded: 2016-09-30T14:00:00.000+00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Novel RNA-Binding Proteins Isolation by the RaPID Methodology at JoVE.com

我们感谢教授杰夫·Gerst和鲍里斯Slobodin在建立快速协议，并提供必要的质粒的有益的建议。我们也感谢Avigail ATIR-兰德博士为她建立从Smoler蛋白质组学中心这个协议和添马舰谢夫博士她与LC-MS / MS分析帮帮忙。我们感谢TG Kinzy教授（罗格斯）为YEF3抗体。这项工作是由来自两国科学基金资助2011013支持。

注意:插入由12 MS2结合位点的序列（MS2循环; MS2L）成所需的基因组基因座，通常是开放读框（ORF）和3&#39;非编码区之间。这种集成的详细协议，其他22提供。验证通过PCR，Northern分析或RT-PCR 20,23正常插入和表达。它以验证该积分不与3&#39;UTR的合成干预是重要的。此外，诱导型启动子（蛋氨酸耗竭）的表达下熔合到SBP的质粒表达MS2-CP也应导入细胞24。相同的菌株，不含所引入的MS12回路，应作为用于检测非特异性信号的控制。 1.快速纯化<ol><li>生长500毫升酵母细胞（使用250-1000毫升的酵母细胞依赖于感兴趣的基因的表达水平），表示MS2标记的mRNA 20和在适当的生长培养基（ 如合成葡萄糖（SD）选择性培养基）1.0在30 摄氏度 - MS2-CP-GFP-SBP融合蛋白24至600 OD 0.8。 </li><li>离心细胞以3000 xg离心在RT 4分钟并弃上清。 </li><li>洗在1×磷酸盐缓冲盐水细胞（PBS）中，离心之前（步骤1.2），重悬在SD等体积（步骤1.1）无蛋氨酸，并孵育45 - 60分钟，在30℃下以诱导MS2的表达-CP-GFP-SBP融合蛋白。 注:较长的诱导时间可能会造成的GFP的聚集。 </li><li>预留1,000萃取池，并用简单的"热酚"的方法，从25这个样本中提取RNA。注意:苯酚是剧毒，应根据其安全指示进行处理。 注:此RNA是孤立的RNA（ 图1A）的质量有很好的借鉴。 </li><li> 500萃取池，1.35毫升37％的甲醛添加到决赛C的0.1％oncentration以交联RNA-蛋白质复合物。在30℃下10分钟继续孵化。注意:甲醛是高毒，应根据其安全指示进行处理。 </li><li>加26.5毫升的2.5M的甘氨酸到0.125微米的最终浓度和孵育3分钟以停止交联。从这个步骤上，雪藏的一切，以尽量减少RNA降解。 </li><li>离心机的细胞在3000 XG 4分钟，4℃，洗用45毫升冷1X PBS。 </li><li>悬浮细胞在每100毫升初始细胞培养1毫升迅速裂解缓冲液。 </li><li>分成500微升等分试样在螺旋盖微量离心管并加入〜400毫克的冷却玻璃珠每个等分试样（大约全0.2毫升PCR管）。 </li><li>裂解的细胞在珠打浆机3分钟。立即把裂解液冰上。 注:细胞裂解释放细胞RNA酶，它们是RNA降解的主要原因。快速裂解液中含有RNA酶INHI高浓度bitors，如非特异性RNA酶抑制剂肝素和特定的抑制剂。还建议迅速工作，藏在心里冷。 </li><li>转移裂解液到新管。皮尔斯在每个离心管的用热针（1.2毫米×40毫米）中底部的小孔，然后将离心管的15ml试管的顶部。使用5ml的注射器的头部作为微量离心管并15毫升管之间的适配器。离心管装配在3000 XG 1分钟，4℃。 </li><li>传送从15毫升管流通到一个新的1.5毫升管和离心机以10,000×g离心10分钟， 在 4℃以清除细胞碎片。 </li><li>池上清液所有1.5毫升管放入一个15毫升管，并预留1/50，并分别RNA和蛋白分离，1/100体积的裂解液。 注意:这些将作为用于随后的分析（ 图1）的"输入"样品。 </li><li>加入抗生物素蛋白300微克每500毫升我的nitial酵母培养和孵化30分钟以恒定滚动（10转）在4℃（这可以减少到10分钟）。 注意:抗生物素蛋白生物素块和生物素化蛋白质从结合到链霉抗珠。 </li><li>与细胞裂解物孵育之前预洗链霉珠。 <ol><li>转约300微升链霉珠浆料到1.5ml微量离心管中，并除去上层清液。这将导致在250微升的珠。用1ml迅速裂解缓冲液洗珠两次。离心链亲和素珠在660 XG在每个步骤之间摄氏四度 2分钟。 </li><li>通过用0.5ml快速裂解缓冲液，0.5毫升10毫克/毫升BSA和10微升10毫克/毫升酵母tRNA的1小时温育阻断珠。珠可以留给O / N在必要时在摄氏 4 度与旋转阻止解决方案。 </li><li>用1ml迅速裂解缓冲液洗两次。 注意:磁珠也可用于减少时间和background。然而，这些具有比琼脂糖珠容量低。 </li></ol></li><li>加入250微升的洗涤的预链霉珠与含抗生物素蛋白裂解物（来自步骤1.14），并在4℃下以恒定旋转（10rpm下）孵育1小时。 注意:此温育时间是提供足够的结合的时间和减少对RNA降解的机会之间的折衷。 </li><li>离心机在4℃下660×g离心2分钟以清除链霉珠并将上清液转移到一个新15ml试管。分别拨出1/50和1/100体积的裂解液RNA和蛋白分离的。 注:这些将是"未绑定"的样本。 </li><li>用1ml迅速裂解缓冲液洗珠，轻轻摇动，转移至新的1.5毫升微量离心管中，并离心分离在步骤1.17。重复此步骤三次。 </li><li>用1ml快速洗涤缓冲液中，并在4℃5分钟此时旋转（10rpm下）洗涤珠。重复此步骤两次。 </l我&gt; <li>与上述1毫升1×PBS中并离心洗涤珠。 </li><li>与120微升1×PBS再次洗珠。离心机如上，并采取RNA和蛋白提取整个上清液作为"洗"样品。 注意:此最后的洗涤样本将指示洗涤的严格性，应该是相同的卷作为洗脱样品。这将简化在随后的分析处理和装载。 </li><li>以洗脱的RNA和RNA相关蛋白从柱中，添加120微升6毫生物素的在1×PBS中并在4℃下以恒定旋转（10rpm下）孵育45分钟。 </li><li>离心机珠如上和洗脱材料转移到一个新的1.5毫升管。再次旋洗脱样品和上层相转移至新的1.5毫升管，以确保没有珠结转。 </li><li>就拿RNA或蛋白质提取样品。 注意:采取的体积应取决于所述RNA或蛋白质的下列测定的表达水平和。作为指导原则，对于北方分析26，取样品的80％，对于Western印迹23,27或RT-PCR 28，取20％，和用于质谱29发现新的蛋白质，取全部样品。 </li></ol> 2. RNA提取<ol><li>加2倍交联逆转缓冲液的等体积并在65℃下孵育45分钟。继续直接提取RNA。 注意:交联逆转缓冲液含有乙二胺四乙酸（EDTA），其显著提高RNA提取20。 </li><li>使用标准的酚:氯仿法26从"输入"和"绑定"的样本（步骤1.13和1.17）提取RNA。 注意:这些样品中含有大量的RNase抑制剂肝素，它可以抑制随后的反向Transcriptase-（RT）利用测定（ 例如 ，RT-PCR），因此，氯化锂沉淀30建议将其删除。该"WASH"和"洗脱"样品（步骤1.21和1.24）含有少量的RNA，并通过以下的步骤中沉淀。 </li><li> 8M胍盐酸（盐酸胍）和100％的乙醇的两卷等体积添加到"洗涤"和"洗脱"样品。孵育在-80℃下为至少2小时。 注意:胍将有效地使蛋白质变性，从而抑制核糖核酸酶和蛋白酶于样品中。 </li><li>离心机以20,000 xg离心，4℃30分钟，并弃去上清液。以20,000 xg离心，4℃15分钟洗一遍用冷80％乙醇，离心沉淀。 </li><li>溶解于400μl无RNA酶的水的RNA沉淀并再次沉淀以除去任何剩余的盐酸胍或其它污染物。添加40微升3M乙酸钠，pH 5.2和800微升的100％乙醇，孵育在-20℃下至少1小时。 </li><li>离心洗按照步骤2.4，并删除所有乙醇10微升提示。重悬RNA佩尔ET的10微升核糖核酸酶自由水。要格外小心为RNA沉淀通常是不可见的。 注意:如在23（ 图1）中所描述的样品可用于Northern分析。 </li></ol> 3.蛋白制品为Western印迹或质谱分析<ol><li>加1/3体积4倍的Laemmli样品缓冲液（LSB）的蛋白质的样品和在65°C孵育45分钟以扭转的RNA和蛋白质的交联。 注:样品可以储存在-20℃。 </li><li>在SDS聚丙烯酰胺凝胶（SDS-PAGE）31运行的蛋白质。根据蛋白质的大小要分析调节凝胶的百分比。 注:10％丙烯酰胺凝胶给出了广泛的分离和良好作为第一近似。 </li><li>转移蛋白的膜作为用于分离效率（ 图 1C）控制的标准免疫印迹分析27。无论使用哪种考马斯亮蓝R染色质谱分析前250或银染。 注意:银染是优选的选择，因为它是更敏感，并且允许更好的检测（ 图1B）。染色可与手动制备溶液或市售试剂盒进行。在最终的染料溶液中的凝胶的温育时间应通过试验确定。潜伏期长可能揭示低丰度蛋白，但可引起高表达的蛋白，如MS2-CP-SBP-GFP是在染色和可能掩盖周围的蛋白质在其附近。此外，则可能出现高背景。认真遵循反应，并加入终止液在适当的时候。 </li><li>从凝胶中切出条带，并将它们传送到1.5毫升管。在-20℃储存的样品，或发送用于蛋白质提取和胰蛋白酶消化，随后用LC-MS / MS分析32。 </li><li>作为替代蛋白质组学方法排除凝胶分离步骤，在这样从步骤1.24消化洗脱材料lution使用胰蛋白酶和受至LC-MS / MS分析33。 注意:省略SDS-PAGE分离简化了协议并增加产量。然而，样品可能包含洗涤剂NP-40，从而抑制质谱分析的踪迹。为了克服这个问题，执行以下步骤: <ol><li> 15分钟-在SDS-PAGE 31 10上运行的蛋白质。 </li><li>切出的车道（ 即 ，其中该蛋白质的位置）的整个部分。 注:蛋白质样品可能包含可能掩盖低丰度蛋白的信号的MS2-CP-SBP蛋白的显著量。 </li></ol></li></ol>

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The authors declare no competing financial interests.

各种方法使用特定的mRNA的分离，以确定它们相关的蛋白质11,34 35。这些方法适用于体外和体内的策略来探测RNA-蛋白的相互作用。 体外方法孵育外生转录的RNA与细胞裂解物来捕获限制性商业惯例和隔离RNP复合物36,37。这种类型的一个有效的方法最近被提出，这使能结合一个调控RNA基序18新蛋白质的鉴定。这些方法的缺点是非特异性限制性?...

RNA结合蛋白（限制性商业惯例）表示S的约10％ 酵母蛋白1,2和哺乳动物蛋白质3-5的约15％。它们被牵连在许多细胞过程如mRNA转录后加工和调节，翻译，核糖体合成，tRNA的氨酰化和修饰，染色质重塑，以及更多。限制性商业做法的一个重要子群mRNA的结合蛋白（mRNPs）6,7。 mRNA中的成熟的过程中，不同的限制性商业惯例结合转录并介导其核加工，出口出来的细胞核，细胞定位，翻译和降解6-8。因此，不同的组在任何时间点绑定到特定的成绩单的限制性商业惯例决定了它的加工和最终的命运。 与基因相关的限制性商业惯例的识别可以显著提高我们的基本的转录后调控过程的了解。不同遗传，微观，生化和生物信息学方法已经用于鉴定参与mRNA调节（在9-11中综述）蛋白。然而，只有少数的这些方法能够与特定的靶mRNA相关的蛋白质的鉴定。值得注意的是，酵母三杂交系统（Y3H），它利用的利益为诱饵mRNA的筛选在酵母细胞中的表达文库。阳性克隆通常是通过培养来选择或记者表达12-14观察。该方法的主要优点是大量可在细胞环境和测量RNA的蛋白质相互作用的强度的能力被扫描的蛋白质。缺点包括相对大量的由于非特异性结合假阳性结果，而对于由于假阴性结果，部分地融合蛋白猎物或诱饵的RNA的错折叠的高电位。 遗传方式的替代方法是亲和力洁净工作台其相关的蛋白的RNA的阳离子。聚A含的mRNA可以通过使用寡聚dT柱进行分离，并且它们的相关的蛋白质通过质谱法进行检测。的RNA-蛋白质相互作用是通过交联的细胞环境，这使得短程共价键是保守的。使用寡聚dT柱产生了与任何含有聚A-mRNA的3,5,15相关的整个蛋白质组的全局视图。但是，这并不能提供与特定的mRNA相关蛋白质的列表。很少方法可用来实现这种识别。该对方法需要核酸与互补性与靶mRNA 16,17的转染。核酸也附连到的肽，其允许交联到限制性商业惯例中紧挨着的相互作用位点。交联后，RBP肽 - 核酸可以分离并进行蛋白质组分析。最近，基于适体的方法是成功地应用到从哺乳动物细胞系18中提取。用链霉改善的亲和力的RNA适体的开发和融合于（在这种情况下，富含AU的元件（ARE））感兴趣的序列。适体 - ARE的RNA附着在链霉抗珠粒并用细胞裂解物混合。与该ARE序列相关联的蛋白质纯化，并通过质谱（MS）来识别。虽然这种方法检测的蜂窝设置（ 即 ， 在体外 ）外面发生关联，则很可能在将来被修改，以便引入适体到基因组中，从而使蛋白质与表达相关的隔离，而在细胞环境（ 即 体内 ）。在酵母中，其中的遗传操作已经非常成熟，快速的方法（以教授杰夫·Gerst的实验室开发）提供了在体内协会19的观点。快速融合了专用性强的MS2外壳蛋白的结合（MS2-CP）的MS2 RNA序列，和的链霉抗结合结构域（SBP）到链霉偶联珠。这使MS2标记mRNA的高效净化通过链霉珠。此外，的MS2环12份表达式允许多达六个MS2-CP的同时结合到RNA和提高其隔离的效率。因此该协议建议，使新颖的mRNA相关蛋白的鉴定一次洗脱的样品通过质谱法进行蛋白组学分析。 我们最近利用快速识别与酵母PMP1 mRNA的20相关联的新的蛋白质。PMP1 mRNA的先前显示与ER膜相关联，并且其3&#39;非翻译区（UTR）被认为是在此关联21的主要决定因素。因此，结合PMP1 3&#39;非编码区限制性商业惯例有可能在其定位可发挥重要的作用。快速接着液相色谱的y质谱/质谱（LC-MS / MS）导致与PMP1 20相互作用的许多新的蛋白质的鉴定。在此，我们提供的快速方法的详细的协议，做需要的重要的控制，这可能提高产量和特异性的技术技巧。

<table><tbody><tr><td>Tris</td><td>sigma</td><td>T1503</td><td></td></tr><tr><td>SDS</td><td>bio-lab</td><td>1981232300</td><td></td></tr><tr><td>DTT</td><td>sigma</td><td>D9779</td><td></td></tr><tr><td>Acidic Phenol (pH 4.3)</td><td>sigma</td><td>P4682</td><td></td></tr><tr><td>Acidic Phenol: Chloroform (5:1, pH 4.3)</td><td>sigma</td><td>P1944</td><td></td></tr><tr><td>Chloroform</td><td>bio-lab</td><td>3080521</td><td></td></tr><tr><td>Formaldehyde</td><td>Frutarom</td><td>5551820</td><td></td></tr><tr><td>Glycine</td><td>sigma</td><td>G7126</td><td></td></tr><tr><td>NP-40</td><td>Calbiochem</td><td>492016</td><td></td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>Phenylmethylsulfonyl Flouride (PMSF)</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>Leupeptin</td><td>Sigma</td><td>L2884</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1153</td><td></td></tr><tr><td>Soybean Trypsin Inhibitor</td><td>Sigma</td><td>T9003</td><td></td></tr><tr><td>Pepstatin</td><td>Sigma</td><td>P5318</td><td></td></tr><tr><td>DNase I</td><td>Promega</td><td>M610A</td><td></td></tr><tr><td>Ribonuclease&amp;nbsp; Inhibitor</td><td>Takara</td><td>2313A</td><td></td></tr><tr><td>Glass Beads</td><td>Sartorius</td><td>BBI-8541701</td><td>0.4-0.6mm diameter&amp;nbsp;</td></tr><tr><td>Mini BeadBeater</td><td>BioSpec</td><td>Mini BeadBeater 16</td><td></td></tr><tr><td>Guanidinium</td><td>Sigma</td><td>G4505</td><td></td></tr><tr><td>Avidin</td><td>Sigma</td><td>A9275</td><td></td></tr><tr><td>Streptavidin Beads</td><td>GE Healthcare&amp;nbsp;</td><td>17-5113-01</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Yeast tRNA</td><td>Sigma</td><td>R8508</td><td></td></tr><tr><td>Biotin</td><td>Sigma</td><td>B4501</td><td></td></tr><tr><td>Yeast extract</td><td>Bacto</td><td>288620</td><td></td></tr><tr><td>peptone</td><td>Bacto</td><td>211677</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>1x Phosphate-Buffered saline (PBS)</td><td></td><td></td><td></td></tr><tr><td>0.2 M NaOH</td><td></td><td></td><td></td></tr><tr><td>4x Laemmli Sample Buffer (LSB)</td><td></td><td></td><td>0.2 M Tris-Hcl pH 6.8, 8% SDS, 0.4 M DTT, 40% glycerol, 0.04% Bromophenol-Blue.</td></tr><tr><td>Hot phenol lysis buffer</td><td></td><td></td><td>10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS&amp;nbsp;</td></tr><tr><td>3 M Sodium Acetate pH 5.2</td><td></td><td></td><td></td></tr><tr><td>100% and 70% Ethanol (EtOH)</td><td></td><td></td><td></td></tr><tr><td>RNase-free water</td><td></td><td></td><td></td></tr><tr><td>RaPID lysis buffer</td><td></td><td></td><td>20 mM Tris pH 7.5, 150 mM NaCl, 1.8 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 0.5% NP-40, 5 mg/ml Heparin, 1 mM Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl Flouride (PMSF), 10 &amp;micro;g/ml Leupeptin, 10 &amp;micro;g/ml Aprotinin, 10 &amp;micro;g/ml Soybean Trypsin Inhibitor, 10 &amp;micro;g/ml Pepstatin, 20 U/ml DNase I, 100 U/ml Ribonuclease&amp;nbsp; Inhibitor.</td></tr><tr><td>2x Cross-linking reversal buffer</td><td></td><td></td><td>100 mM Tris pH 7.4, 10 mM EDTA, 20 mM DTT, 2 % SDS.</td></tr><tr><td>RaPID wash buffer</td><td></td><td></td><td>20 mM Tris-HCl pH 7.5,&amp;nbsp; 300 mM NaCl, 0.5% NP-40</td></tr><tr><td>0.5 M EDTA pH 8</td><td></td><td></td><td></td></tr><tr><td>Silver Stain Plus Kit</td><td>Bio-Rad&amp;nbsp;</td><td>161-0449</td><td>For detecting proteins in polyacrylamide gels</td></tr><tr><td>SD selective medium&amp;nbsp;</td><td></td><td></td><td>1.7 g/l Yeast nitrogen base with out amino acids and ammonium sulfate, 5 g/l Ammonium sulfate, 2% glucose, 350 mg/l Threonine, 40 mg/l Methionine, 40 mg/l Adenine, 50 mg/l Lysine, 50 mg/l Tryptophan, 20 mg/l Histidine, 80 mg/l Leucine, 30 mg/l Tyrosine, 40 mg/l Arginine</td></tr><tr><td>Anti-eEF3 (EF3A,YEF3)</td><td>Gift from Kinzy TG. (UMDNJ Robert Wood Johnson Medical School)</td><td>1:5,000</td><td></td></tr><tr><td>Anti GFP antibody</td><td>Santa Cruz</td><td>sc-8334</td><td>1:3,000</td></tr><tr><td>Anti rabbit IgG-HRP conjugated</td><td>SIGMA</td><td>A9169</td><td>1:10,000</td></tr></tbody></table>

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.

迅速使与其相关的蛋白质的特定的靶RNA的分离。临界其成功是保持所述RNA完整尽可能，由此获得的蛋白质的足够量。为了确定RNA的分离效率和质量，Northern分析进行（ 图1A）。 Northern分析具有直接向快速的效率和质量的优点。因此，全长和降解产物的相对量可以在单次运行来确定。核糖体RNA（个rRNA）是在溴化乙锭染色容易地检测，和洗脱样品中的缺少个rRNA的?...

Watch this Scientific Journal Video about Novel RNA-Binding Proteins Isolation by the RaPID Methodology at JoVE.com

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

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 ...

novel-rna-binding-proteins-isolation-by-the-rapid-methodology

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Genetics

8.9K 次观看.  Technion - Israel Institute of Technology. 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. 

视频: Novel RNA-Binding Proteins Isolation by the RaPID Methodology

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.

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. ...

科研

生物化学

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 ...

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

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 ...

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 ...

遗传学

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 ...

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

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 ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

RNA-Protein Pull-Down Assay to Isolate RNA-Binding Proteins via Affinity Extraction

Source: Kresoja-Rakic, J., et al. <a href="https://app.jove.com/v/57516">Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells.</a> J. Vis. Exp. (2018)
This video demonstrates an in vitro RNA pull-down assay to identify RNA-binding proteins (RBPs), which interact with the adenylate-uridylate-rich element (ARE) sequences in mRNA. The target RBPs from a cell lysate are mixed with an RNA probe to form RNA-protein complexes. The complexes are isolated via affinity purification utilizing the affinity of the desthiobiotin label of the RNA probe to a streptavidin-labeled magnetic bead.

通过亲和提取分离 RNA 结合蛋白的 RNA-蛋白质沉降测定

Source: Kresoja-Rakic, J., et al. Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells. J. Vis. ...