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
Summary
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.
Abstract
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.
Introduction
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'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5'5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'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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Dynabeads Protein A | Invitrogen | 10002D | |
| Dynabeads Protein G | Invitrogen | 10004D | |
| Anti- FLAG antibody | Sigma | F3165 | |
| Anti- FLAG antibody | Sigma | F7425 | |
| Anti-RHA antibody | Vaxron | PA-001 | |
| TRizol LS reagent | Life technology | 10296-028 | |
| RNase H | Ambion | AM2292 | |
| Chloroform | Fisher Scientific | BP1145-1 | |
| Isopropanol | Fisher Scientific | BP26184 | |
| RNaeasy clean-up column | Qiagen | 74204 | |
| Omniscript reverse transcriptase | Qiagen | 205113 | |
| RNase Out | Invitrogen | 10777-019 | |
| Protease inhibitor cocktail | Roche | 5056489001 | |
| Triton X-100 | Sigma | X100 | |
| NP-40 | Sigma | 98379 | |
| Glycerol | Fisher Scientific | 17904 | |
| Random hexamer primers | Invitrogen | N8080127 | |
| Oligo-dT primers | Invitrogen | AM5730G | |
| PCR primers | IDT | Gene specific primers for PCR amplification | |
| Oligonucleotide for RNase H mediated cleavage | IDT | Anti-sense primer for target RNA | |
| Trypsin | Gibco Life technology | 25300-054 | |
| DMEM tissue culture medium | Gibco Life technology | 11965-092 | |
| Fetal bovine serum | Gibco Life technology | 10082-147 | |
| Tris base | Fisher Scientific | BP152-5 | |
| Sodium chloride | Fisher Scientific | S642-212 | |
| Magnesium chloride | Fisher Scientific | BP214 | |
| DTT | Fisher Scientific | R0862 | |
| Sucrose | Fisher Scientific | BP220-212 | |
| Nitrocellulose membrane | Bio-Rad | 1620112 | |
| Magnetic stand | 1.7 ml micro-centrifuge tube holding | ||
| Laminar hood | For animal tissue culture | ||
| CO2 incubator | For animal tissue culture | ||
| Protein gel apparatus | Protein sample separation | ||
| Protein transfer apparatus | Protein sample transfer | ||
| Ready to use protein gels (4-15%) | Protein sample separation | ||
| Table top centrifuge | Pellet down the sample | ||
| Table top rotator | Mix the sample end to end | ||
| Vortex | Mix the samples |
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