前体mRNA底物的3'末端切割：用无细胞体系中的RNA加工反应分析<em>在体外</em
Summary
RNA聚合酶II合成延伸超过所述成熟mRNA的3'末端的前体RNA。的成熟RNA的末端cotranscriptionally生成的，在由RNA序列决定的一个部位，通过裂解复合物的内切核酸酶的活性。在这里，我们详细研究裂解反应在体外的方法。
Abstract
哺乳动物mRNA的3'末端不被由RNA聚合酶II（RNPII）突然终止转录的形成。相反，RNPII合成前体的mRNA之外的成熟RNA的末端，内切酶活性的主动过程，需要在特定的位点。前体RNA切割的CA二核苷酸后通常会发生10-30个核苷酸下游的共识polyA位点（AAUAAA）。从裂解复合物，约800 kDa的多因子蛋白复合物，蛋白质完成这个特定的核酸酶活性。特定的RNA序列的上游和多聚A位点的下游控制裂解复合物的募集。紧接在切割后，mRNA前由聚腺苷酸聚合酶（PAP）多聚腺苷酸化，以产生成熟的稳定的RNA消息。
一种RNA转录物的3'端的处理可以使用移动的核提取物与特定的放射性标记的RNA底物进行研究。总之，长32 </ SUP> P-标记的未裂解的前体RNA被孵育体外核提取物和裂解是通过凝胶电泳和放射自显影评估。当适当的切割发生在较短的5'裂解产物进行检测和定量。在这里，我们描述了裂解分析法详细使用，作为一个例子，对HIV-1的mRNA的3'末端加工。
Introduction
最成熟的真核生物的RNA消息（mRNA的）的生物合成需要几个转录后修饰，如封盖，剪接和多聚腺苷酸化。这些修饰通常耦合，以确保正确的处理1，并强烈增加mRNA的稳定性。
由聚（A）聚合酶（PAP）2-4 3'哺乳动物预mRNA的末端形成由新生RNA，随后加入腺苷酸残基到5的内切核苷酸裂解产生'裂解产物。在哺乳动物中，断裂是由多组分蛋白复合物完成约800 kDa的，是组装在特定的预RNA序列。所述聚（A）信号序列，一个高度保守的典型六核苷酸序列AAUAAA，引导切割位点在约10-30个核苷酸的下游。这个网站是专门由切割和多聚腺苷酸化特异性因子（CPSF）及73 kD的亚基确认CSPF包含内切核酸酶的活性。裂解刺激因子（CSTF）结合更加堕落GU-或U丰富的元素序列中的poly（A）位点下游。还需要裂解是哺乳动物裂解因子I（CFIM）和哺乳动物裂解因子II（CFII）。 CFIM结合上游序列中的元素（使用）的具体UGUA（N）的网站，已经被定义为一些基因，似乎参与重要的生理过程5-8。
在体外实验中 ，RNA加工反应通过使用放射性标记的RNA底物9-12常用分析。这些可以通过从噬菌体启动子的T7或SP6失控转录来合成。当研究一种多聚腺苷酸化位点尚未之前其特征在于，它需要使用的基因组DNA，而不是基因产生的RNA底物，作为重要的下游序列中可能不存在于cDNA的。设计底物，包括至少150个核苷酸upstrea米和50从上成熟的mRNA切割位点/结束nt的下游。裂解产物迁移比基材快;然而，因为其他片段可以通过非特异性核酸酶作用而生成的，该反应的特异性具有由其上的正确处理的信号序列依赖性进行验证。因此，RNA底物具有在AAUAAA序列（ 例如，AAGAAA）的点突变可作为阴性对照的裂解反应。
由于少量的放射性标记的RNA被用于裂解反应中，存在于高丰度在大多数核提取核糖核酸酶可以是有问题的，并限制对提取物的制备起始材料的选择范围。 HeLa细胞中往往含有内源性核糖核酸酶水平低，因而在这些测定中表现良好。
在RNA底物在体内和体外的信使核糖核酸是紧接着的poly（A）另外，周四S中的裂解的中间不存在可检测量。因此，研究一个特定的RNA序列或涉及的裂解反应的蛋白质，实验是在防止多聚腺苷酸化的发生条件进行。有乳沟的多聚腺苷酸化无依赖性，反之亦然，所以人能阻止多聚腺苷酸化而不伤害裂解反应。因此，ATP被替换为缺少3'羟基基团，以便只有一个单核苷酸可以在多聚（A）位点被结合并可以被检测到刚切割的RNA链终止类似物。
由于复杂性和这种类型的检测的特殊性程度高，我们描述了一个详细的视频协议由前体mRNA的体外切割/聚（A）机械研究核酸内切裂解。我们描述了如何准备主管核提取物，产生放射性标记的RNA底物，进行裂解反应，并分析和解释结果ING产品。 图1显示了HIV-1的前mRNA的3'末端也可以用于裂解分析法进行编码的RNA底物的例子。艾滋病毒RNA基因组的3'端是由许多重要的调控序列，如聚（A）位点，一个G+ü丰富的地区，以及使用的元素，这些都是必要的病毒mRNA转录13高效成熟。在这个例子中，我们所期望的输入RNA底物为338 nt的，一经裂解237 nt的。如果多聚腺苷酸化被允许发生，产品一抹黑将237和437个核苷酸之间被观察到。
Protocol
Representative Results
Discussion
体外前体mRNA 3'裂解反应，在HeLa细胞核提取物或从这些提取物分馏裂解因素进行，使识别的核心裂解因素，其主要复合物16-21。许多与这些因素相关的更多的蛋白质最近已确定了22个，并在体外的反应可能会继续阐明这些蛋白是如何有助于反应轻。也许是因为在体内的反应似乎是cotranscriptional 23，或者是因为一些影响因素可能提取和透析过程中丢失时…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SV是感谢来自美国国立卫生研究院K22AI077353和Landenberger基金会的资助。 KR感激来自美国国立卫生研究院（5SC1GM083754）承认资金。
Materials
| 1L Celstir Flask | Wheaton | 356884 | Different sizes available |
| 4 Position Slow Speed Stirrer | VWR | 12621-076 | |
| Swinging Bucket Centrifuge | Beckman Coulter | Allegra x-15R with SX4500 Rotor | |
| Ultra Centrifuge | Beckman Coulter | Optima L-100 XP Ultra with SW41 Rotor | |
| Table Top Centrifuge 5417R | Eppendorf | Refridgerated | |
| Thermomixer incubator | Eppendorf | ||
| 250ml Conicle Tubes | Corning | 430776 | |
| 50ml Conicle Tubes | BD Falcon | 352098 | |
| Ultra-clear Centrifuge Tubes | Beckman Coulter | 344059 | |
| JOKLIK Modified MEM | Lonza | 04-719Q | |
| Fetal Bovine Serum | Atlas | F-0500-A | Heat inactivated |
| L-Glut:Pen:Strep | Gemini Bio-Products | 400-110 | |
| 1M Tris-HCl pH 8.0 | Mediatech | 46-031-CM | |
| Magnesium Chloride | Fisher | BP214-500 | |
| Potassium Chloride | MP Biomedicals | 194844 | |
| HEPES | Fisher | BP310-500 | |
| DTT | Alexis Biomedicals | 280-001-G-010 | |
| Glycerol | Fisher | BP229-4 | |
| 5M Sodium Chloride Solution | Mediatech | 46-032-CV | |
| EDTA 0.5M Solution | Sigma-Aldrich | E7889-100ml | |
| EDTA | Fisher | BP120-500 | |
| PMSF | Thermo Scientific | 36978 | |
| Ammonium Sulfate | Fisher | A702-500 | |
| cOmplete EDTA-Free Protease inhibitor cocktail tablets | Roche | 04 693 132 001 | |
| Slide-A-Lyzer Dialysis Cassettes Kit | Thermo Scientific | 66372 | MWCO 7000 0.5ml-3ml |
| 15ml Dounce Tissue grinder set | Sigma-Aldrich | D9938-1SET | Different sizes available |
| Expand High Fidelity PCR Kit | Roche | 11 732 650 001 | |
| 10mM dNTP Mix | Invitrogen | Y02256 | 10mM each nucleotide |
| MaxiScript SP6/T7 Kit | Ambion | AM1322 | |
| m7G(5')ppp(5') G RNA Cap | New England Biolabs | S1404S | |
| Century Marker Template Plus | Ambion | AM7782 | |
| Easytides UTP [alpha-32p]-250uCi | Perkin Elmer | BLU507H250UC | |
| Gel loading buffer II | Ambion | 8546G | |
| DEPC treated water | Ambion | AM9906 | |
| 10x TAE | Fisher | BP13354 | |
| 10x TBE | Ameresco Life Sciences | 0658-4L | |
| 10x PBS | Fisher | BP399-20 | |
| Urea | Fisher | BP169-212 | |
| Ammonium Persulfate | Bio-Rad | 161-0700 | |
| TEMED | Fisher | BP150-20 | |
| Ammonium Acetate | Fisher | A637-500 | |
| 40% 19:1 Acrylamide:Bis-acrylamide | Bio-Rad | 161-0144 | |
| Glycogen | Roche | 10 901 393 001 | |
| 100% Absolute Ethanol 200 Proof | Acros | 61509-0040 | |
| Acid Phenol-Chloroform | Ambion | 9720 | For RNA |
| Scintilation Fluid | Fisher | SX18-4 | |
| Rnase Inhibitor | Promega | N261B | |
| Poly (Vinyl alcohol) PVA | Sigma-Aldrich | P8136-250G | |
| Creatine Phosphate | Calbiochem | 2380 | |
| 100mM dATP | Fisher | BP2560-4 | |
| SDS | Acros | 23042-5000 | |
| Proteinase K | Fisher | BP1700-100 | |
| Adjustable Sequencing Unit | Sigma-Aldrich | Z351881-1EA | |
| Binder Clips | Office Depot | 838-056 | |
| 20cmx42cm glass plates | Sigma-Aldrich | Z352543 | 1SET |
| 20cmx22cm glass plates | Sigma-Aldrich | Z35252-7 | 1SET |
| 20cmx42cm Aluminum Cooling Plates | Sigma-Aldrich | Z352667 | 1EA |
| 0.4mmx22cm Spacers | Sigma-Aldrich | Z35230-6 | 1SET |
| 0.4mmx42cm Spacers | Sigma-Aldrich | Z352314-1 | 1SET |
| 8-well Comb | Sigma-Aldrich | Z35195-4 | 1EA |
| 16-well Comb | Sigma-Aldrich | Z351962 | 1EA |
| 32-well Comb | Sigma-Aldrich | Z351970 | 1EA |
| Gel Repel Coating | C.B.S. Scientific | SGR-0401 | or SGR-0101 for individual bottle |
| Gel Loading Tips | Rainin | GT-10-4 | 0.1-10uL |
| Sequencing PowerPac HV | Bio-Rad | PowerPac HV | 5000V/500mA/400W |
| Gel Dryer Model 583 | Bio-Rad | Model 583 | |
| Hydrotech Vacuum Pump for gel dryer | Bio-Rad | ||
| Glogos II Glow-in-the-dark Markers | Agilent | 420201 | |
| Film 8×10 | Midsci | BX810 | |
| Film 14×17 | Phenix | F-BX1417 | |
| Autoradiography Cassette | Fisher | FBCA 1417 | 8×10 size available |
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