的快速方法新颖的RNA结合蛋白隔离
Summary
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.
Abstract
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.
Introduction
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'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'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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Tris | sigma | T1503 | |
| SDS | bio-lab | 1981232300 | |
| DTT | sigma | D9779 | |
| Acidic Phenol (pH 4.3) | sigma | P4682 | |
| Acidic Phenol: Chloroform (5:1, pH 4.3) | sigma | P1944 | |
| Chloroform | bio-lab | 3080521 | |
| Formaldehyde | Frutarom | 5551820 | |
| Glycine | sigma | G7126 | |
| NP-40 | Calbiochem | 492016 | |
| Heparin | Sigma | H3393 | |
| Phenylmethylsulfonyl Flouride (PMSF) | Sigma | P7626 | |
| Leupeptin | Sigma | L2884 | |
| Aprotinin | Sigma | A1153 | |
| Soybean Trypsin Inhibitor | Sigma | T9003 | |
| Pepstatin | Sigma | P5318 | |
| DNase I | Promega | M610A | |
| Ribonuclease Inhibitor | Takara | 2313A | |
| Glass Beads | Sartorius | BBI-8541701 | 0.4-0.6mm diameter |
| Mini BeadBeater | BioSpec | Mini BeadBeater 16 | |
| Guanidinium | Sigma | G4505 | |
| Avidin | Sigma | A9275 | |
| Streptavidin Beads | GE Healthcare | 17-5113-01 | |
| Bovine serum albumin (BSA) | Sigma | A7906 | |
| Yeast tRNA | Sigma | R8508 | |
| Biotin | Sigma | B4501 | |
| Yeast extract | Bacto | 288620 | |
| peptone | Bacto | 211677 | |
| Glucose | Sigma | G8270 | |
| 1 x Phosphate-Buffered saline (PBS) | |||
| 0.2 M NaOH | |||
| 4 x Laemmli Sample Buffer (LSB) | 0.2 M Tris-Hcl pH 6.8, 8% SDS, 0.4 M DTT, 40% glycerol, 0.04% Bromophenol-Blue. | ||
| Hot phenol lysis buffer | 10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS | ||
| 3 M Sodium Acetate pH 5.2 | |||
| 100% and 70% Ethanol (EtOH) | |||
| RNase-free water | |||
| RaPID lysis buffer | 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 µg/ml Leupeptin, 10 µg/ml Aprotinin, 10 µg/ml Soybean Trypsin Inhibitor, 10 µg/ml Pepstatin, 20 U/ml DNase I, 100 U/ml Ribonuclease Inhibitor. | ||
| 2x Cross-linking reversal buffer | 100 mM Tris pH 7.4, 10 mM EDTA, 20 mM DTT, 2 % SDS. | ||
| RaPID wash buffer | 20 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.5% NP-40 | ||
| 0.5 M EDTA pH 8 | |||
| Silver Stain Plus Kit | Bio-Rad | 161-0449 | For detecting proteins in polyacrylamide gels |
| SD selective medium | 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 | ||
| Anti-eEF3 (EF3A,YEF3) | Gift from Kinzy TG. (UMDNJ Robert Wood Johnson Medical School) | 1:5,000 | |
| Anti GFP antibody | Santa Cruz | sc-8334 | 1:3,000 |
| Anti rabbit IgG-HRP conjugated | SIGMA | A9169 | 1:10,000 |
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