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 border="0" cellpadding="0" cellspacing="0" width="632">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Tris</td>
			<td style="width:107px;">sigma</td>
			<td style="width:119px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td>bio-lab</td>
			<td align="right">1981232300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acidic Phenol (pH 4.3)</td>
			<td>sigma</td>
			<td>P4682</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acidic Phenol: Chloroform (5:1, pH 4.3)</td>
			<td>sigma</td>
			<td>P1944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>bio-lab</td>
			<td align="right">3080521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Frutarom</td>
			<td align="right">5551820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>sigma</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td>Calbiochem</td>
			<td align="right">492016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethylsulfonyl Flouride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepstatin</td>
			<td>Sigma</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Promega</td>
			<td>M610A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ribonuclease&#160; Inhibitor</td>
			<td>Takara</td>
			<td>2313A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Beads</td>
			<td>Sartorius</td>
			<td>BBI-8541701</td>
			<td>0.4-0.6mm diameter&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini BeadBeater</td>
			<td>BioSpec</td>
			<td colspan="2">Mini BeadBeater 16</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidinium</td>
			<td>Sigma</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Avidin</td>
			<td>Sigma</td>
			<td>A9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Beads</td>
			<td>GE Healthcare&#160;</td>
			<td>17-5113-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast tRNA</td>
			<td>Sigma</td>
			<td>R8508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin</td>
			<td>Sigma</td>
			<td>B4501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td>Bacto</td>
			<td dir="LTR">288620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">peptone</td>
			<td>Bacto</td>
			<td dir="LTR">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 x Phosphate-Buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 M NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 x 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 height="21">
			<td height="21" style="height:21px;">Hot phenol lysis buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 M Sodium Acetate pH 5.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% and 70% Ethanol (EtOH)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RaPID lysis buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Tris pH 7.5, 150 mM NaCl, 1.8 mM MgCl2, 0.5% NP-40, 5 mg/ml Heparin, 1 mM Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl Flouride (PMSF), 10 &#181;g/ml Leupeptin, 10 &#181;g/ml Aprotinin, 10 &#181;g/ml Soybean Trypsin Inhibitor, 10 &#181;g/ml Pepstatin, 20 U/ml DNase I, 100 U/ml Ribonuclease&#160; Inhibitor.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2x Cross-linking reversal buffer</td>
			<td></td>
			<td></td>
			<td dir="LTR">100 mM Tris pH 7.4, 10 mM EDTA, 20 mM DTT, 2 % SDS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RaPID wash buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Tris-HCl pH 7.5,&#160; 300 mM NaCl, 0.5% NP-40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 M EDTA pH 8</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silver Stain Plus Kit</td>
			<td>Bio-Rad&#160;</td>
			<td>161-0449</td>
			<td>For detecting proteins in polyacrylamide gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SD selective medium&#160;</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 height="21">
			<td height="21" style="height:21px;">Anti-eEF3 (EF3A,YEF3)</td>
			<td colspan="2">Gift from Kinzy TG. (UMDNJ Robert Wood Johnson Medical School)</td>
			<td>1:5,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti GFP antibody</td>
			<td>Santa Cruz</td>
			<td>sc-8334</td>
			<td>1:3,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

Research

JoVE Journal

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

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

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

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 ...

科研

遗传学

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

滞后链DNA合成的动力学研究<em&gt;体外</em&gt;由T7噬菌体复制蛋白

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Novel Sequence Discovery by Subtractive Genomics

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

The purpose of this protocol is to use a combination of computational and bench research to find novel sequences that cannot be easily separated from a co-purifying sequence, which may be only partially known.

减法基因组学的新序列发现

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

纳米测序全血 mRNA 测序

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

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 ...

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

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

探索序列空间,识别调节RNA结合蛋白的结合位点

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